Bmx mediated signal transduction in irradiated vascular endothelium

ABSTRACT

Provided are methods for modulating the proliferation of cells and tissues. In some embodiments, the methods include administering to a subject an effective amount of a modulator of a biological activity of a bone marrow X kinase (Bmx) gene product. Also provided are methods for increasing the radiosensitivity of a target cell or tissue, methods for suppressing tumor growth, methods for inhibiting tumor blood vessel growth, and compositions that include modulators of a biological activity of a bone marrow X kinase (Bmx) gene product.

CROSS REFERENCE TO RELATED APPLICATION

The presently disclosed subject matter claims the benefit of U.S.Provisional Patent Application Ser. No. 60/997,124, filed Oct. 1, 2007;the disclosure of which is incorporated herein by reference in itsentirety

GOVERNMENT INTEREST

The presently disclosed subject matter was made with U.S. Governmentsupport under Grant No. 2R01-CA89674-04 awarded by the NationalInstitutes of Health/National Cancer Institute. Thus, the U.S.Government has certain rights in the presently disclosed subject matter.

TECHNICAL FIELD

The presently disclosed subject matter relates to methods andcompositions for modulating cellular and/or tissue proliferation. Insome embodiments, the compositions comprise a modulator of a Bmx geneproduct biological activity. In some embodiments, the methods compriseadministering a composition comprising a modulator of a Bmx gene productbiological activity to a subject in order to modulate cellular or tissueproliferation.

BACKGROUND

Ionizing radiation is useful in the treatment of cancer and for ablationof pathologic tissues because of the cytotoxic effects which result frompersistent DNA double strand breaks or activation of program cell death(Haimovitz-Friedman et al., 1994; Garcia-Barros et al., 2003; Brown &Attardi, 2005). Radiation causes rapidly proliferating cells, such astumor and cancer cells, to undergo cell death by apoptosis, both in vivoand in vitro (Antonakopoulos et al., 1994; Li et al., 1994; Mesner etal., 1997).

Current radiation therapy is frequently unsuccessful at completelyeradicating cancer cells from a patient, however. This is true for atleast two reasons. One reason cancer can recur is that it is often notpossible to deliver a sufficiently high dose of local radiation to killtumor cells without concurrently creating an unacceptably high risk ofdamage to the surrounding normal tissue. Another reason is that tumorsshow widely varying susceptibilities to radiation-induced cell death.Ionizing radiation activates pro-survival response throughphosphoinositide 3-kinase/Akt (PI3K/Akt) and mitogen-activated proteinkinase (MAPK) signal transduction pathways (Dent et al., 2003; Tan &Hallahan, 2003; Tan et al., 2006; Yacoub et al., 2006). PI3K catalyzesthe addition of a phosphate group to the inositol ring ofphosphoinositides normally present in the plasma membrane of cells(Wymann & Pirola, 1998). The products of these reactions, includingphosphatidyl-4,5-bisphosphate and phosphatidyl-3,4,5-trisphosphate, arepotent second messengers of several RTK signals (Cantley, 2002). Invitro studies have indicated that PI3K and Akt are involved in growthfactor-mediated survival of various cell types (Datta et al., 1999),including neuronal cells (Yao & Cooper, 1995; Dudek et al., 1997; Weiner& Chun, 1999), fibroblasts (Kauffmann-Zeh et al., 1997; Fang et al.,2000), and certain cells of hematopoietic origin (Katoh et al., 1995;Kelley et al., 1999; Somervaille et al., 2001).

Another obstacle to designing effective radiotherapy is that there is apoor correlation between cellular responses to ionizing radiation invitro and in vivo. For example, glioblastoma multiforme (GBM) isinsensitive to radiation treatment, and has a universally fatal clinicaloutcome in both children and adults (Walker et al., 1980; Wallner etal., 1989; Packer, 1999). In vitro studies, however, show that human GBMcell lines exhibit radiosensitivity that is similar to that seen in celllines derived from more curable human tumors (Allam et al., 1993;Taghian et al., 1993). In accord with the clinical data, the use of invivo animal models has shown that GBM tumors in vivo are much moreradioresistant than the cell lines used to produce them are in vitro(Baumann et al., 1992; Allam et al., 1993; Taghian et al., 1993; Advaniet al., 1998; Staba et al., 1998). Thus, the inability to predict theradiosensitivity of a tumor in vivo based upon in vitro experimentationcontinues to be a significant obstruction to the successful design ofradiotherapy treatments of human cancers.

Tumor cells could show enhanced radiosensitivity in vitro compared to invivo due to the absence of an angiogenic support network in vitro, thepresence of which appears to contribute to a tumor's radioresistance invivo. The response of tumor microvasculature to radiation is dependentupon the dose and time interval after treatment (Kallman et al., 1972;Song et al., 1972; Hilmas & Gillette, 1975; Johnson, 1976; Yamaura etal., 1976; Ting et al., 1991). Tumor blood flow decreases when highdoses of radiation in the range of 20 Grays (Gy) to 45 Gy are used (Songet al., 1972). In contrast, blood flow increases when relatively lowradiation doses, for example below 500 rads, are administered (Kallmanet al., 1972; Hilmas & Gillette, 1975; Johnson, 1976; Yamaura et al.,1976; Gorski et al., 1999). In irradiated mouse sarcomas, for example,blood flow increased during the 3 to 7 days immediately followingirradiation (Kallman et al., 1972). Thus, the microvasculature mightserve to protect tumor cells from radiation-induced cell death.

Thus, there exists an ongoing and long-felt need in the art foreffective therapies for enhancing the efficacy of radiotherapy,particularly in the context of tumors that are resistant toradiotherapy. To address this need, the presently disclosed subjectmatter provides inter alia methods for increasing the radiosensitivityof a cell or tissue. Such methods can be useful for enhancing theefficacy of anti-proliferative treatments such as, but not limited tochemotherapy and radiotherapy, among other applications.

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently disclosed subject matter provides methods for modulatingproliferation of a cell or of a tissue in a subject. In someembodiments, the methods comprise administering to the subject aneffective amount of a modulator of a biological activity of a bonemarrow X kinase (Bmx) gene product. In some embodiments, the cell is atumor cell or a vascular endothelial cell. In some embodiments, thesubject is a mammal. In some embodiments, the Bmx gene product isencoded by a naturally occurring nucleic acid sequence that is at least95% identical to nucleotides 174-2198 of SEQ ID NO: 1 or is encoded by anaturally occurring nucleic acid sequence that is at least 95% identicalto nucleotides 112-2136 of SEQ ID NO: 3. In some embodiments, themodulator is an inhibitor of a biological activity of a Bmx geneproduct. In some embodiments, the inhibitor is selected from the groupconsisting of (2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide,an antibody that specifically binds to the Bmx gene product to inhibit abiological activity of the Bmx gene product, and a nucleic acid thatinhibits a biological activity of the Bmx gene product by RNAinterference. In some embodiments, the nucleic acid that inhibits abiological activity of the Bmx gene product by RNA interferencecomprises a short interfering RNA (siRNA) or a short hairpin RNA (shRNA)that targets a Bmx gene product encoded by a nucleic acid sequencecomprising nucleotides 174-2198 of SEQ ID NO: 1 or nucleotides 112-2136of SEQ ID NO: 3. In some embodiments, the siRNA or the shRNA is encodedby a recombinant virus and the administering comprises administering aneffective amount of the recombinant virus to the subject to modulateproliferation of a cell or of a tissue in the subject.

The presently disclosed subject matter also provides methods forincreasing the radiosensitivity of a target cell or tissue. In someembodiments, the methods comprise contacting the target cell or tissuewith an effective amount of a modulator of a biological activity of abone marrow X kinase (Bmx) gene product. In some embodiments, themodulator of a biological activity of a Bmx gene product comprises abone marrow X kinase (Bmx) antagonist, a vector encoding a bone marrow Xkinase (Bmx) antagonist, or a combination thereof. In some embodiments,the target cell or tissue comprises an endothelial cell or endothelialtissue. In some embodiments, the endothelial tissue is vascularendothelium. In some embodiments, the target cell or tissue is a tumorcell or a tumor. In some embodiments, the tumor comprises a radiationresistant tumor. In some embodiments, the target cell or tissuecomprises vasculature supplying blood flow to a tumor. In someembodiments, the subject is a mammal. In some embodiments, theadministering a bone marrow X kinase (Bmx) antagonist comprisesadministering a minimally therapeutic dose of a Bmx antagonist. In someembodiments, the administering comprises administering a compositioncomprising a bone marrow X kinase (Bmx) antagonist, a vector encoding abone marrow X kinase (Bmx) antagonist, or a combination thereof and apharmaceutically acceptable carrier. In some embodiments, the bonemarrow X kinase (Bmx) antagonist is selected from the group consistingof (2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, anantibody that specifically binds to the Bmx gene product to inhibit abiological activity of the Bmx gene product, and a nucleic acid thatinhibits a biological activity of the Bmx gene product by RNAinterference. In some embodiments, the bone marrow X kinase (Bmx)antagonist comprises a small interfering RNA (siRNA) targeted to a Bmxgene product.

The presently disclosed subject matter also provides methods forsuppressing tumor growth in a subject. In some embodiments, the methodscomprise administering to the subject an effective amount of a modulatorof a biological activity of a bone marrow X kinase (Bmx) gene productand treating the tumor with ionizing radiation, whereby tumor growth issuppressed. In some embodiments, the subject is a mammal. In someembodiments, the administering comprises administering a minimallytherapeutic dose of the modulator. In some embodiments, theadministering comprises administering a composition comprising a bonemarrow X kinase (Bmx) antagonist, a vector encoding a bone marrow Xkinase (Bmx) antagonist, or a combination thereof and a pharmaceuticallyacceptable carrier. In some embodiments, the modulator is selected fromthe group consisting of(2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, an antibodythat specifically binds to the Bmx gene product to inhibit a biologicalactivity of the Bmx gene product, and a nucleic acid that inhibits abiological activity of the Bmx gene product by RNA interference. In someembodiments, the modulator comprises a small interfering RNA (siRNA)targeted to a Bmx gene product. In some embodiments, the tumor comprisesa radiation resistant tumor. In some embodiments, the treating the tumorwith ionizing radiation comprises treating the tumor with asubtherapeutic dose of ionizing radiation.

The presently disclosed subject matter also provides methods forinhibiting tumor blood vessel growth. In some embodiments, the methodscomprise administering to the subject an effective amount of a modulatorof a biological activity of a bone marrow X kinase (Bmx) gene productand treating the tumor with ionizing radiation, whereby tumor bloodvessel growth is inhibited. In some embodiments, the administeringcomprises administering a minimally therapeutic dose of the modulator.In some embodiments, the modulator comprises a composition comprising abone marrow X kinase (Bmx) antagonist, a vector encoding a bone marrow Xkinase (Bmx) antagonist, or a combination thereof and a pharmaceuticallyacceptable carrier. In some embodiments, the modulator is selected fromthe group consisting of(2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, an antibodythat specifically binds to the Bmx gene product to inhibit a biologicalactivity of the Bmx gene product, and a nucleic acid that inhibits abiological activity of the Bmx gene product by RNA interference. In someembodiments, the modulator comprises a small interfering RNA (siRNA)targeted to a Bmx gene product. In some embodiments, the subject is amammal. In some embodiments, the tumor comprises a radiation resistanttumor. In some embodiments, the treating the tumor with ionizingradiation comprises treating the tumor with a subtherapeutic dose ofionizing radiation. In some embodiments, the methods further comprisereducing the vascular length density of the tumor blood vessels.

The presently disclosed subject matter also provides methods forinhibiting a condition associated with undesirable angiogenesis in asubject. In some embodiments, the methods comprise administering to thesubject an effective amount of a bone marrow X kinase (Bmx) antagonist.In some embodiments, the condition associated with undesirableangiogenesis is selected from the group consisting of a cancer, a tumor,macular degeneration, and endometriosis. In some embodiments, the Bmxantagonist is selected from the group consisting of(2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, an antibodythat specifically binds to the Bmx gene product to inhibit a biologicalactivity of the Bmx gene product, and a nucleic acid that inhibits abiological activity of the Bmx gene product by RNA interference. In someembodiments, the nucleic acid that inhibits a biological activity of theBmx gene product by RNA interference comprises a small interfering RNA(siRNA) targeted to a Bmx gene product. In some embodiments, the subjectis a mammal.

The presently disclosed subject matter also provides expressionconstructs encoding a short interfering RNA (siRNA) or a short hairpinRNA (shRNA) that modulates expression of a Bmx gene product. In someembodiments, the Bmx gene product is encoded by a naturally occurringnucleic acid sequence that is at least 95% identical to nucleotides174-2198 of SEQ ID NO: 1 or nucleotides 112-2136 of SEQ ID NO: 3, is anaturally occurring non-human ortholog thereof, or a naturally occurringsplice variant or allelic variant of any of these. In some embodiments,the naturally occurring non-human ortholog thereof comprises an aminoacid sequence that is at least 95% identical to an amino acid sequenceas set forth in any of even numbered SEQ ID NOs: 2-20 or is encoded by anaturally occurring nucleic acid sequence that is at least 95% identicalto an open reading frame of any of odd numbered SEQ ID NOs: 1-19.

Thus, it is an object of the presently disclosed subject matter toprovide methods for modulating proliferation of a cell or of a tissue ina subject.

An object of the presently disclosed subject matter having been statedhereinabove, and which is achieved in whole or in part by the presentlydisclosed subject matter, other objects will become evident as thedescription proceeds when taken in connection with the accompanyingdrawings as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B present the results of experiments showing that Bmx isactivated by radiation in endothelial cells. Human umbilical veinendothelial cells (HUVEC) were either sham irradiated or radiated with 2Gy.

FIG. 1A is a Western blot with an antibody directed againstphosphotyrosine of total lysates from cells after incubation at 37° C.for the indicated times in minutes. Western blots are shown ofphosphor-tyrosine 40 (PY40) Bmx, indicative of activation, as well astotal Bmx and actin for normalization.

FIG. 1B depicts the results of in vitro kinase assays (IVK) in which Bmxwas immunoprecipitated (IP:Bmx) from the lysates as in FIG. 1A andeluted under non-denaturing conditions. Following elution, kinase assaybuffer was added for 20 minutes. Samples were separated by SDS-PAGE.Western blot analysis using anti-phosphotyrosine (IB:PY) antibody wasused to detect autophosphorylation.

FIGS. 2A and 2B present the results of experiments showing retroviralshRNA knockdown of Bmx in HUVEC. Multiple shRNA retroviral plasmidconstructs (Bmx constructs A-E, and negative control construct; Neg)were transfected into LiNX cells to produce retroviral supernatants asdescribed in Materials and Methods for the EXAMPLES hereinbelow. HUVECwere infected with the retroviral supernatants and incubated for 48hours.

FIG. 2A depicts Western blot analysis using an anti-Bmx antibody todetect total Bmx levels from total protein lysates of the transfectedcells that had been separated by SDS-PAGE and transferred to a solidsupport.

FIG. 2B is a bar graph showing the results of MTT-based survival assays.Cells infected with either Bmx or control shRNA were incubated for 48hours prior to plating of 10,000 cells/well in 96-well dishes. Cellswere treated with either 0 or 2 Gy and incubated for 24 hours. Cellswere then treated with WST-1 reagent and incubated for 2 hours prior todye quantification at 450 nm (OD₄₅₀). Normalized mean absorbance valueswith standard errors are shown. Shaded bars—negative control; stippledbars—Bmx shRNA.

FIGS. 3A-3D depict the results of experiments showing thatradiation-induced endothelial cell cytotoxicity is enhanced by Bmxinhibition.

FIG. 3A shows the results of in vitro kinase assays (IVK) for LFM-A13.Bmx was immunoprecipitated (IP:Bmx) from lysates of HUVEC pre-treatedwith either 30 μM LFM-A13 or DMSO vehicle control for 45 minutes priorto 3 Gy irradiation. IP:Bmx samples were then eluted in non-denaturingconditions and subjected to kinase assay. Samples were then loaded forSDS-PAGE and anti-phosphotyrosine (IB:PY) Western blotting.

FIG. 3B is a graph showing the results of clonogenic assays of HUVECcells with 1 hour pre-incubation with 30 μM LFM-A13 or DMSO vehiclecontrol. Cells were counted and plated and subjected to the indicateddoses of radiation and colonies formed over 10 days. Surviving colonieswere plotted as a function of cells plated and normalized by the platingefficiency for each condition. Standard error (SE) bars are shown.

FIG. 3C shows the results of analysis of apoptosis in cells treated with30 μM LFM-A13 or DMSO vehicle alone (negative control). LFM-A13 or DMSOvehicle was added to plated cells with or without 3 Gy radiation (IR).After 24 hours, cells were trypsinized and collected for flow cytometryusing Annexin V/Propidium iodide staining. The first five (5) panels area series of FACS scatter plots showing the percent of cells undergoingearly (Q4) and late (Q2) apoptosis compared to viable cells (Q3) anddead cells (Q1). The sixth panel is a plot of the quantification ofearly+late apoptosis as mean and standard error. *: p<0.001 vs. LFM-A13or 3 Gy alone.

FIG. 3D depicts apoptosis in cells preincubated with 30 μM LFM-A13 orDMSO vehicle control treated with either 3 or 6 Gy of irradiation andincubated at 37° C. for 24 hours prior to fixing and staining with DAPI.The left panel is a fluorescence micrograph of such cells, and the rightpanel is a bar graph of the percent of cells demonstrating apoptoticmorphology shown as mean and standard error. *: p<0.05 vs. control. **:p=0.001 vs. LFM-A13 alone.

FIGS. 4A-4D depict the results of experiments showing that endothelialcell function is attenuated by Bmx inhibition and radiation.

FIG. 4A shows the results of endothelial cell closure assays in which80% confluent HUVEC were subjected to a gap formation using a 200 μlpipette tip. Cells were then treated with 30 μM LFM-A13 or DMSO vehiclecontrol for one hour followed by 3 Gy. 12 and 24 hours later, cells werefixed with 70% ethanol and stained with methylene blue. Shown arerepresentative photographs.

FIG. 4B is a bar graph of the mean and standard error of relative celldensity in the original gap area (n=4). *: p<0.05 vs. control. **:p<0.01 vs. LFM-A13 alone.

FIG. 4C depicts a micrograph of HUVEC placed onto MATRIGEL™ plugs andtreated with either 30 μM LFM-A13 or DMSO vehicle control followed by 3Gy irradiation.

FIG. 4D is a bar graph of the mean number of tubules of the cells shownin FIG. 4C with standard error bars. The cells depicted in FIG. 4C werefixed and tubules were quantitated by NIH ImageJ software. *: p<0.05 vs.control. **: p<0.005 vs. LFM-A13 or 3 Gy alone.

FIGS. 5A-5D depict the results of experiments showing that tumorvascular destruction is enhanced by Bmx inhibition and radiation. In thetumor vascular window model, a transparent window chamber was placedonto the dorsal skin fold of C57BL/6 mice to allow for visualization ofblood vessels. LLC cells were injected into the chamber and once vesselsformed (6-8 days), the mice were treated with one intraperitoneal doseof 50 mg/kg LFM-A13 or DMSO vehicle control followed one hour followedby 2 Gy. Microscopic photos were taken daily and the density of bloodvessels was quantified for each treatment group.

FIG. 5A are representative photographs and FIG. 5B is a bar graph of themean vascular length density and standard error (n=3) for each treatmentgroup. *: p<0.0014 vs. LFM-A13 or 3 Gy alone. Lewis lung carcinoma cellswere implanted into the hind limb of mice. Once tumors formed, the micewere treated with five consecutive daily treatments of 50 mg/kg LFM-A13(+LFM-A13) or DMSO control (−LFM-A13) and/or 3 Gy fractions (XRT).Tumors were harvested and stained for anti-CD34.

FIG. 5C is a set of photomicrographs of immunohistochemistry, and FIG.5D is a bar graph of the mean level of CD34 staining and standard errorcalculated for each treatment condition. **: p=0.043 vs. IR. *: p=0.0001vs. LFM-A13 or vehicle control.

FIGS. 6A and 6B are graphs showing that Bmx inhibition enhancesradiation efficacy in a xenograft lung cancer model. For FIG. 6A, LLCswere treated with DMSO or LFM-A13 prior to 0, 2, 4, or 6 Gy irradiationfor clonogenic survival assay as performed with HUVEC in FIGS. 2 and 3.Surviving colonies were plotted as a fraction of cells plated that wasnormalized by the plating efficiency for each condition. SE Bars areshown. For FIG. 6B, LLCs were injected into the hind limb of C57BL/6mice and tumors were allowed to form. Animals were separated into 4treatment groups, DMSO vehicle control with sham irradiation (C),LFM-A13 with sham irradiation (L), radiation alone (X), or combinedLFM-A13 with radiation (L+X). Treatments were given as i.p. injection of50 mg/kg LFM-A13 45 min prior to 3 Gy which was given daily for 5consecutive days. Tumor size was measured and volume was calculated andmean tumor volume and standard error for each group was plotted. Tumorgrowth delay was determined for 1.5 cm³ volume time point for eachgroup. p=0.027 for combination treatment group.

FIG. 7 is a depiction of an exemplary structure for an siRNA targeted toa Bmx gene product.

FIG. 8 is an alignment of amino acid sequences of Bmx gene products fromvarious species listed in Table 1. Each amino acid sequence wastruncated at the N-terminus (if necessary) so that amino acid positionnumber 1 of the human BMX corresponded to the first amino acid of eachBmx gene product. Above each set of ten amino acid sequences are symbolsdenoting the degree of conservation among the sequences. *—all sequencesinclude the same amino acid. :—high degree of conservation. .—moderateconservation. [no symbol]—lower or no conservation.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1 and 2 are a nucleotide and amino acid sequences,respectively, for a human Bmx gene product, transcript variant 1(GENBANK® Accession Nos. NM_(—)203281 and NP_(—)975010, respectively).

SEQ ID NOs: 3 and 4 are a nucleotide and amino acid sequences,respectively, for a human Bmx gene product, transcript variant 2(GENBANK® Accession Nos. NM_(—)001721 and NP_(—)001712, respectively).

SEQ ID NOs: 5 and 6 are a nucleotide and amino acid sequences,respectively, for a Macaca mulatta Bmx gene product, transcript variant1 (GENBANK® Accession Nos. XM_(—)001101349 and XP_(—)001101349,respectively).

SEQ ID NOs: 7 and 8 are a nucleotide and amino acid sequences,respectively, for a Macaca mulatta Bmx gene product, transcript variant2 (GENBANK® Accession Nos. XM_(—)001101166 and XP_(—)001101166,respectively).

SEQ ID NOs: 9 and 10 are a nucleotide and amino acid sequences,respectively, for a Macaca mulatta Bmx gene product, transcript variant3 (GENBANK® Accession Nos. XM_(—)001101250 and XP_(—)001101250,respectively).

SEQ ID NOs: 11 and 12 are a nucleotide and amino acid sequences,respectively, for a murine Bmx gene product (GENBANK® Accession Nos.NM_(—)009759 and NP_(—)033889, respectively).

SEQ ID NOs: 13 and 14 are a nucleotide and amino acid sequences,respectively, for a rat Bmx gene product (GENBANK® Accession Nos.NM_(—)001109016 and NP_(—)001102486, respectively).

SEQ ID NOs: 15 and 16 are a nucleotide and amino acid sequences,respectively, for a bovine Bmx gene product (GENBANK® Accession Nos.XM_(—)610012 and XP_(—)610012, respectively).

SEQ ID NOs: 17 and 18 are a nucleotide and amino acid sequences,respectively, for a canine Bmx gene product (GENBANK® Accession Nos.XM_(—)548870 and XP_(—)548870, respectively).

SEQ ID NOs: 19 and 20 are a nucleotide and amino acid sequences,respectively, for an Equus caballus Bmx gene product (GENBANK®AccessionNos. XM_(—)001490091 and XP_(—)001490141, respectively).

DETAILED DESCRIPTION I. General Considerations

The microvasculature is a major component of cancer and supports tumorgrowth. Several groups have studied the inherent resistance of tumorvascular endothelium to cytotoxic effects of ionizing radiation (IR). IRactivates signal transduction through the phosphatidyl inositol-3 kinase(PI3K)/Akt pathway, which enhances endothelial cell viability (Valerieet al., 2007; Sonveaux et al., 2007; Zingg et al., 2004). It haspreviously been shown that IR induced Akt activation is eliminated byover-expression of mutant p85 component of PI3K (Tan & Hallahan, 2003).Mutant p85 functions as a dominant negative by preventing activation ofp110 catalytic subunit of PI3K. This inhibition prevents the productionof phosphatidylinositol phosphates (PIPs) that activate Akt resulting inenhanced radiation effect. Therefore, inhibition of the PI3K signaltransduction pathway can abrogate the endothelial cell survivalsignaling mediated by Akt.

Although the PI3K/Akt pathway is a major contributor to radiationresistance seen in tumor microvasculature, other pathways activatedshortly after IR are also being investigated. Indeed, the activation ofAkt has been shown to be critically dependent on binding of Akt'spleckstrin homology (PH) domain to specific PIPs, PIP3 in particular,that allows the co-localization of Akt with upstream activators (Chan etal., 1999). Bone marrow X kinase (Bmx), also known as epithelial andendothelial tyrosine kinase (Etk), contains a PH domain as well as Srchomology-2 (SH2) and −3 (SH3) domains capable of interacting withseveral types of second messengers and adaptor proteins that are knownto be present in human umbilical vein endothelial cells (HUVEC; Qiu &Kung, 2000; Chen et al., 2001; Smith et al., 2001; Pan et al., 2002;Vargas et al., 2002; Yang et al., 2002; Nore et al., 2003). Bmx is theubiquitously expressed member of the Tec family of non-receptor tyrosinekinases with high expression in lung, prostate, and the heart (Qiu &Kung, 2000; Smith et al., 2001). In addition, salivary epithelium,granulomonocytic cells, endothelial cells and epithelial cells expressthis protein in relatively high amounts (Kaukonen et al., 1996; Mano,1999; Wen et al.; 1999; Qiu & Kung, 2000; Smith et al., 2001). Bmxappears to act both upstream and downstream of PI3K (Qiu et al., 1998;Ekman et al., 2000; Qiu & Kung, 2000; Chen et al., 2001; Smith et al.,2001; Chau et al., 2002; Zhang et al., 2003; Chau et al., 2005). Bmxalso interacts with G-proteins (Qiu & Kung, 2000; Lee et al., 2001; Kimet al., 2002; Cote et al., 2005), integrins/NRTK's (Chen et al., 2001:Abassi et al., 2003), tumor necrosis factor receptors (Pan et al.,2002), as well as various protein tyrosine phosphatases (Jui et al.,2000) and lipid phosphatases (Tomlinson et al., 2004).

Disclosed herein are investigations into Bmx signaling within thevascular endothelium. As set forth in more detail herein below, Bmx wasactivated rapidly in response to clinically relevant doses of ionizingradiation. Bmx inhibition enhanced the efficacy of radiotherapy inendothelial cells, tumor vascular endothelium in lung cancer tumors inmice. Retroviral shRNA knockdown of Bmx protein enhanced HUVECradiosensitization. Furthermore, pretreatment of HUVEC with apharmacological inhibitor of Bmx, LFM-A13, produced significantradiosensitization of endothelial cells as measured by clonogenicsurvival analysis and apoptosis as well as functional assays includingcell migration and tubule formation. In vivo, LFM-A13, when combinedwith radiation resulted in significant tumor microvascular destructionas well as enhanced tumor growth delay. Bmx therefore represents amolecular target for the development of novel radiosensitizing agents.

II. Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and“the” mean “one or more” when used in this application, including theclaims. Thus, the phrase “a cell” refers to one or more cells, and canthus also refer to a tissue or an organ.

The term “about”, as used herein when referring to a measurable valuesuch as an amount of weight, time, dose (e.g., radiation dose), etc. ismeant to encompass in some embodiments variations of ±20% or ±10%, insome embodiments ±5%, in some embodiments ±1%, in some embodiments ±01%,and in some embodiments ±0.01% from the specified amount, as suchvariations are appropriate to perform the disclosed methods.

The terms “nucleic acid molecule” and “nucleic acid” each refer todeoxyribonucleotides or ribonucleotides and polymers thereof insingle-stranded, double-stranded, or triplexed form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides that have similar properties as the referencenatural nucleic acid. The terms “nucleic acid molecule” and “nucleicacid” can also be used in place of “gene”, “cDNA”, or “mRNA”. Nucleicacids of the presently claimed subject matter can be cloned,synthesized, recombinantly altered, mutagenized, or combinationsthereof. Standard recombinant DNA and molecular cloning techniques usedto isolate nucleic acids are known in the art. Site-specific mutagenesisto create base pair changes, deletions, or small insertions is alsoknown in the art as exemplified by publications. See e.g., Sambrook &Russell, 2001; Silhavy et al., 1984; Glover & Hames, 1995; and Ausubel,1995. Nucleic acids can be derived from any source, including anyorganism.

The term “substantially identical”, as used herein to describe a degreeof similarity between nucleotide sequences, refers to two or moresequences that have in some embodiments at least about least 60%, inanother embodiment at least about 70%, in another embodiment at leastabout 80%, in another embodiment about 90% to about 99%, in anotherembodiment about 95% to about 99%, and in some embodiments about 99%nucleotide identity, when compared and aligned for maximumcorrespondence, as measured using a sequence comparison algorithm or byvisual inspection. In some embodiments, the substantial identity existsin nucleotide sequences of at least about 100 residues, in someembodiments in nucleotide sequences of at least about 150 residues, andin some embodiments in nucleotide sequences comprising a full lengthcoding sequence.

In some embodiments, substantially identical sequences can comprisepolymorphic sequences. The term “polymorphic” refers to the occurrenceof two or more genetically determined alternative sequences or allelesin a population. An allelic difference can be as small as one base pair.In some embodiments, substantially identical sequences can comprisemutagenized sequences, including sequences comprising silent mutations.A mutation can comprise a single base change.

Another indication that two nucleotide sequences are substantiallyidentical is that the two molecules specifically or substantiallyhybridize to each other under stringent conditions. In the context ofnucleic acid hybridization, two nucleic acid sequences being comparedcan be designated a “probe” and a “target”. A “probe” is a referencenucleic acid molecule, and a “target” is a test nucleic acid molecule,often found within a heterogeneous population of nucleic acid molecules.A “target sequence” is synonymous with a “test sequence”.

In some embodiments, a nucleotide sequence employed for hybridizationstudies or assays includes probe sequences that are complementary to ormimic at least an about 14 to 40 nucleotide sequence of a nucleic acidmolecule of the presently claimed subject matter. In some embodiments,probes comprise 14 to 20 nucleotides, or even longer where desired, suchas 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the fulllength of any one of the sequences of the presently claimed subjectmatter. Such probes can be readily prepared by, for example, chemicalsynthesis of the fragment, by application of nucleic acid amplificationtechnology, or by introducing selected sequences into recombinantvectors for recombinant production.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent hybridization and wash conditions when thatsequence is present in a complex nucleic acid mixture (e.g., totalcellular DNA or RNA).

The phrase “hybridizing substantially to” refers to complementaryhybridization between a probe nucleic acid molecule and a target nucleicacid molecule and embraces minor mismatches that can be accommodated byreducing the stringency of the hybridization media to achieve thedesired hybridization.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern blot analysis are both sequence- andenvironment-dependent. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, 1993. Generally, highly stringent hybridization andwash conditions are selected to be about 5° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. Typically, under “stringent conditions” a probe willhybridize specifically to its target subsequence, but to no othersequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor Southern or Northern Blot analysis of complementary nucleic acidshaving more than about 100 complementary residues is overnighthybridization in 50% formamide with 1 mg of heparin at 42° C. An exampleof highly stringent wash conditions is 15 minutes in 0.1×SSC at 65° C.An example of stringent wash conditions is 15 minutes in 0.2×SSC bufferat 65° C. See Sambrook & Russell, 2001 for a description of SSC buffer.Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example of medium stringency washconditions for a duplex of more than about 100 nucleotides is 15 minutesin 1×SSC at 45° C. An example of low stringency wash for a duplex ofmore than about 100 nucleotides is 15 minutes in 4× to 6×SSC at 40° C.For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1MNa⁺ ion, typically about 0.01 to 1M Na⁺ ion concentration (or othersalts) at pH 7.0-8.3, and the temperature is typically at least about30° C. Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2-fold (or higher) than that observed for an unrelated probe inthe particular hybridization assay indicates detection of a specifichybridization.

The following are additional examples of hybridization and washconditions that can be used to identify nucleotide sequences that aresubstantially identical to reference nucleotide sequences of thepresently claimed subject matter: a probe nucleotide sequence in oneexample hybridizes to a target nucleotide sequence in 7% sodium dodecylsulfate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C. followed by washing in2×SSC, 0.1% SDS at 50° C.; in another example, a probe and targetsequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO₄, 1 mMEDTA at 50° C. followed by washing in 1×SSC, 0.1% SDS at 50° C.; inanother example, a probe and target sequence hybridize in 7% sodiumdodecyl sulfate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C. followed bywashing in 0.5×SSC, 0.1% SDS at 50° C.; in another example, a probe andtarget sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5MNaPO₄, 1 mM EDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at50° C.; in another example, a probe and target sequence hybridize in 7%sodium dodecyl sulfate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C. followedby washing in 0.1×SSC, 0.1% SDS at 65° C.

A further indication that two nucleic acid sequences are substantiallyidentical is that the proteins encoded by the nucleic acids aresubstantially identical, share an overall three-dimensional structure,or are biologically functional equivalents. Nucleic acid molecules thatdo not hybridize to each other under stringent conditions are stillsubstantially identical if the corresponding proteins are substantiallyidentical. This can occur, for example, when two nucleotide sequencesare significantly degenerate as permitted by the genetic code.

The term “conservatively substituted variants” refers to nucleic acidsequences having degenerate codon substitutions wherein the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Ohtsuka et al., 1985; Batzer etal., 1991; Rossolini et al., 1994).

In making biologically functional equivalent amino acid substitutions,the hydropathic index of amino acids can be considered. Each amino acidhas been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics, these are: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art. See e.g., Kyte & Doolittle, 1982. It is known that certainamino acids can be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, oneexample involves the substitution of amino acids whose hydropathicindices are within ±2 of the original value, another example involvesthose that are within ±1 of the original value, and yet another exampleinvolves those within ±0.5 of the original value.

It is also understood in the art that the substitution of like aminoacids can be made effectively based on hydrophilicity. U.S. Pat. No.4,554,101 describes that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with its immunogenicity and antigenicity, e.g., with abiological property of the protein. It is understood that an amino acidcan be substituted for another having a similar hydrophilicity value andstill obtain a biologically equivalent protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, one exampleinvolves the substitution of amino acids whose hydrophilicity values arewithin ±2 of the original value, another example involves those that arewithin ±1 of the original value, and still another example involvesthose within ±0.5 of the original value.

The term “subsequence” refers to a sequence of nucleic acids thatcomprises a part of a longer nucleic acid sequence. An exemplarysubsequence is a probe, described herein above, or a primer. The term“primer” as used herein refers to a contiguous sequence comprising insome embodiments about 8 or more deoxyribonucleotides orribonucleotides, in some embodiments 10-20 nucleotides, and in someembodiments 20-30 nucleotides of a selected nucleic acid molecule. Theprimers of the presently claimed subject matter encompassoligonucleotides of sufficient length and appropriate sequence so as toprovide initiation of polymerization on a nucleic acid molecule of thepresently claimed subject matter. In the context of an siRNA (discussedin more detail hereinbelow), an exemplary subsequence is a sequence thatcomprises part of a duplexed region of an siRNA, one strand of which iscomplementary to the sequence of an mRNA.

III. Radiosensitivity

In some embodiments, a method for increasing the radiosensitivity of atarget tissue in a subject via administration of a modulator of abiological activity of a bone marrow X kinase (Bmx) gene product isprovided. The method comprises administering a Bmx antagonist to thesubject, whereby the radiosensitivity of a target tissue is increased.The presently described subject matter also provides a method forsuppressing tumor growth in a subject. The method comprises: (a)administering a modulator of a biological activity of a bone marrow Xkinase (Bmx) gene product to a subject bearing a tumor to increase theradiosensitivity of the tumor; and (b) treating the tumor with ionizingradiation, whereby tumor growth is suppressed. The presently claimedsubject matter also provides a method for inhibiting tumor blood vesselgrowth. The method comprises (a) administering modulator of a biologicalactivity of a bone marrow X kinase (Bmx) gene product to a subjectbearing a tumor to increase the radiosensitivity of tumor blood vessels;and (b) treating the tumor with ionizing radiation, whereby tumor bloodvessel growth is inhibited.

The term “radiosensitivity” as used herein to describe a target tissuerefers to a quality of susceptibility to treatment using ionizingradiation. This susceptibility can result from direct effects of theradiation on the cells of the target tissue themselves. For example,radiation can cause the cells of the target tissue to undergo apoptosisas a result of either DNA damage or another cell autonomous mechanism.Alternatively, radiosensitivity can result from indirect effects, suchas effects on the microenvironment of the cells of the target tissue,for example, on the blood vessels supplying nutrients and oxygen to thetarget tissue. Thus, radiotherapy can be used to suppress the growth ofa radiosensitive target tissue.

Radiosensitivity can be quantified by determining a minimal amount ofionizing radiation that can be used to delay target tissue growth. Thus,the term “radiosensitivity” refers to a quantitative range of radiationsusceptibility.

The term “target tissue” refers to any cell or group of cells present ina subject. This term includes single cells and populations of cells. Theterm includes but is not limited to cell populations comprising glandsand organs such as skin, liver, heart, kidney, brain, pancreas, lung,stomach, and reproductive organs. It also includes but is not limited tomixed cell populations such as bone marrow. Further, it includes but isnot limited to such abnormal cells as neoplastic or tumor cells, whetherindividually or as a part of solid or metastatic tumors. The term“target tissue” as used herein additionally refers to an intended sitefor accumulation of a modulator of a biological activity of a bonemarrow X kinase (Bmx) gene product following administration to asubject. For example, the methods of the presently claimed subjectmatter employ a target tissue comprising a tumor and/or the vasculatureproviding oxygen to a tumor.

The term “suppressing tumor growth” refers to an increase in a durationof time required for a tumor to grow a specified amount. For example,treatment can extend the time required for a tumor to increase in volumeto 2-fold, 3-fold, 4-fold, 5-fold, or longer relative to an initial dayof measurement (day 0) or the time required to grow to a volume of 1cm³.

The terms “radiation resistant tumor” and “radioresistant tumor” eachgenerally refer to a tumor that is substantially unresponsive toradiotherapy when compared to other tumors. Representative radiationresistant tumor models include but are not limited to glioblastomamultiforme and melanoma.

The term “increase” as used herein to refer to a change inradiosensitivity of a tumor refers to change that renders a tumor moresusceptible to destruction by ionizing radiation. Alternatively stated,an increase in radiosensitivity refers to a decrease in the minimalamount of ionizing radiation that effectively suppresses tumor growth.An increase in radiosensitivity can also comprise suppressed tumorgrowth or inhibited tumor blood vessel growth when a modulator of abiological activity of a bone marrow X kinase (Bmx) gene product isadministered with radiation as compared to a same dose of radiationalone. An increase in radiosensitivity refers to an increase of in someembodiments at least about 2-fold, in some embodiments at least about5-fold, and in some embodiments at least 10-fold. In some embodiments ofthe presently claimed subject matter, an increase in radiosensitivitycomprises a transformation of a radioresistant tumor to a radiosensitivetumor.

The methods of the presently claimed subject matter are useful forincreasing the radiosensitivity of a target tissue, for suppressingtumor growth, and/or for inhibiting blood vessel growth in any subject.Thus, the term “subject” as used herein includes any vertebrate species,for example, warm-blooded vertebrates such as mammals and birds. Moreparticularly, the methods of the presently claimed subject matter areprovided for the treatment of tumors in mammals such as humans, as wellas those mammals of importance due to being endangered (such as Siberiantigers), of economic importance (animals raised on farms for consumptionby humans) and/or social importance (animals kept as pets or in zoos) tohumans, for instance, carnivores other than humans (such as cats anddogs), swine (pigs, hogs, and wild boars), ruminants and livestock (suchas cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also provided is the treatment of birds, including those kindsof birds that are endangered or kept in zoos, as well as fowl, and moreparticularly domesticated fowl or poultry, such as turkeys, chickens,ducks, geese, guinea fowl, and the like, as they are also of economicimportance to humans.

The term “tumor” as used herein encompasses both primary andmetastasized solid tumors and carcinomas of any tissue in a subject,including but not limited to breast; colon; rectum; lung; oropharynx;hypopharynx; esophagus; stomach; pancreas; liver; gallbladder; bileducts; small intestine; urinary tract including kidney, bladder andurothelium; female genital tract including cervix, uterus, ovaries(e.g., choriocarcinoma and gestational trophoblastic disease); malegenital tract including prostate, seminal vesicles, testes and germ celltumors; endocrine glands including thyroid, adrenal, and pituitary; skin(e.g., hemangiomas and melanomas), bone or soft tissues; blood vessels(e.g., Kaposi's sarcoma); brain, nerves, eyes, and meninges (e.g.,astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,neuroblastomas, Schwannomas and meningiomas). The term “tumor” alsoencompasses solid tumors arising from hematopoietic malignancies such asleukemias, including chloromas, plasmacytomas, plaques and tumors ofmycosis fungoides and cutaneous T-cell lymphoma/leukemia, and lymphomasincluding both Hodgkin's and non-Hodgkin's lymphomas. The term “tumor”also encompasses radioresistant tumors, including radioresistantvariants of the any of the tumor listed above.

IV. Modulators of Bmx Gene Product Biological Activities

Any suitable modulator of a biological activity of a Bmx gene productcan be used in accordance with the methods of the presently claimedsubject matter. In some embodiments, the antagonist has a capacity toincrease the radiosensitivity of a target tissue. In some embodiments, amodulator of a biological activity of a Bmx gene product also showsanti-angiogenic activity, angiostatic activity, or both.

The phrase “a modulator of a biological activity of a Bmx gene product”as used herein can refer to a molecule or other chemical entity having acapacity for specifically binding to a Bmx gene product to therebyenhance or suppress a biological activity of the Bmx gene product.Modulators of biological activities of Bmx gene products include but arenot limited to small molecule inhibitors, neutralizing antibodies, andnucleic acid-based antagonists (e.g., siRNAs directed against a Bmx geneproducts).

The term “Bmx” refers to a bone marrow X kinase (Gene Symbol BMX), whichis a nonreceptor tyrosine kinase gene of the BTK/ITK/TEC/TXK family. Bmxnucleotide and amino acid sequences from several species have beendetermined, a non-limiting subset of which are set forth in Table 1. Analignment of the amino acid sequences presented in even-numbered SEQ IDNOs: 2-20 that has been maximized for overlap beginning at amino acidposition 1 of SEQ ID NO: 2 is presented in FIG. 8.

TABLE 1 Bmx GENBANK ® Sequences Nucleic Acid Amino Acid OrganismAccession No. Accession No. Homo sapiens NM_203281; NP_975010; SEQ IDNO: 1 SEQ ID NO: 2 NM_001721; NP_001712; SEQ ID NO: 3 SEQ ID NO: 4Macaca mulatta XM_001101349; XP_001101349; SEQ ID NO: 5 SEQ ID NO: 6XM_001101166; XP_001101166; SEQ ID NO: 7 SEQ ID NO: 8 XM_001101250;XP_001101250; SEQ ID NO: 9 SEQ ID NO: 10 Mus musculus NM_009759;NP_033889; SEQ ID NO: 11 SEQ ID NO: 12 Rattus norvegicus NM_001109016;NP_001102486; SEQ ID NO: 13 SEQ ID NO: 14 Bos taurus XM_610012;XP_610012; SEQ ID NO: 15 SEQ ID NO: 16 Canis familiaris XM_548870;XP_548870; SEQ ID NO: 17 SEQ ID NO: 18 Equus caballus XM_001490091;XP_001490141; SEQ ID NO: 19 SEQ ID NO: 20

The phrase “open reading frame” (ORF) refers to a sequence ofnucleotides that are translated into a polypeptide. An open readingframe generally begins with an initiator codon (ATG/AUG) and terminateswith the translation termination codon (e.g., TAG/UAG, TAA/UAA, orTGA/UGA) also referred to as a “stop codon”. Table 2 summarizes thenucleotides that correspond to the open reading frames of the Bmx geneproducts listed in Table 1.

TABLE 2 Bmx Gene Product Open Reading Frames Organism SEQ ID NO: ORFNucleotides Homo sapiens SEQ ID NO: 1 174-2201 SEQ ID NO: 3 112-2139Macaca mulatta SEQ ID NO: 5 162-2201 SEQ ID NO: 7 187-2229 SEQ ID NO: 9143-2170 Mus musculus SEQ ID NO: 11 156-2111 Rattus norvegicus SEQ IDNO: 13 139-2106 Bos taurus SEQ ID NO: 15   1-1965 Canis familiaris SEQID NO: 17   1-2628 Equus caballus SEQ ID NO: 19   1-1965

The term “binding” refers to an affinity between two molecules, forexample, an inhibitor and a target molecule. As used herein, the phrases“specific binding” and “selective binding” refer to a preferentialbinding of one molecule for another in a mixture of molecules. Thebinding of an inhibitor to a target molecule generally can be consideredspecific or selective if the binding affinity is about 1×10⁴ M⁻¹ toabout 1×10⁶ M⁻¹ or greater.

IV.A. Small Molecules

The term “small molecule” as used herein refers to a compound, forexample an organic compound, with a molecular weight of in someembodiments less than about 1,000 daltons, in some embodiments less thanabout 750 daltons, in some embodiments less than about 600 daltons, andin some embodiments less than about 500 daltons. A small molecule alsohas a computed log octanol—water partition coefficient in the range ofabout −4 to about +14 in some embodiments, and in the range of about −2to about +7.5 in some embodiments.

A representative, non-limiting example of a modulator of a biologicalactivity of a Bmx gene product is LFM-A13, which has the chemical names(2Z)-2-cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide andα-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide. Itsmolecular formula is C₁₁H₈Br₂N₂O₂, and it has a molecular weight of360.00. It is available from Sigma-Aldrich Chemical Co. of St. Louis,Mo., United States of America.

IV.B. Anti-Bmx Antibodies

The presently claimed subject matter further provides a modulator of abiological activity of a Bmx gene product that comprises an antibodythat specifically binds a Bmx gene product. Optionally, a modulator of abiological activity of a Bmx gene product can further comprise a carrierfor sustained bioavailability of the antibody at a tumor. The disclosureherein provides that a prolonged or sustained release of modulator of abiological activity of a Bmx gene product is optionally employed toenhance the therapeutic effect of combined Bmx modulation and radiation.

The term “antibody” indicates an immunoglobulin protein, or functionalportion thereof, including a polyclonal antibody, a monoclonal antibody,a chimeric antibody, a hybrid antibody, a single chain antibody (e.g., asingle chain antibody represented in a phage library), a mutagenizedantibody, a humanized antibody, and antibody fragments that comprise anantigen binding site (e.g., Fab and Fv antibody fragments). In someembodiments, an antibody of the presently claimed subject matter is amonoclonal antibody.

Techniques for preparing and characterizing antibodies are known in theart. See e.g., Harlow & Lane, 1988 and U.S. Pat. Nos. 4,196,265;4,946,778; 5,091,513; 5,132,405; 5,260,203; 5,677,427; 5,892,019;5,985,279; 6,054,561. Single chain antibodies can be identified byscreening a phage antibody library, for example as described by U.S.Pat. Nos. 6,174,708; 6,057,098; 5,922,254; 5,840,479; 5,780,225;5,702,892; and 5,667,988.

An antibody of the presently claimed subject matter can further bemutagenized or otherwise modified to improve antigen binding and/orantibody stability. For example, to prevent undesirable disulfide bondformation, a nucleotide sequence encoding the variable domain of anantibody or antibody fragment can be modified to eliminate at least oneof each pair of codons that encode cysteines for disulfide bondformation. Recombinant expression of the modified nucleotide sequence,for example in a prokaryotic expression system, results in an antibodyhaving improved stability. See U.S. Pat. No. 5,854,027.

IV.C. Aptamers

In some embodiments, a biological activity of a Bmx gene productcomprises an aptamer that specifically binds to a Bmx polypeptide. Asused herein, “aptamer” refers in general to either an oligonucleotide ofa single defined sequence or a mixture of said oligonucleotides, whereinthe mixture retains the properties of binding specifically to the targetmolecule. Thus, as used herein “aptamer” denotes both singular andplural sequences of oligonucleotides, as defined hereinabove. The term“aptamer” is meant to refer to a single- or double-stranded nucleic acidwhich is capable of binding to a protein or other molecule, and therebydisturbing the protein's or other molecule's function.

In general, aptamers comprise in some embodiments about 10 to about 100nucleotides, in some embodiments about 15 to about 40 nucleotides, insome embodiments about 20 to about 40 nucleotides, in thatoligonucleotides of a length that falls within these ranges are readilyprepared by conventional techniques. Optionally, aptamers can furthercomprise in some embodiments a minimum of approximately 6 nucleotides,in some embodiments 10 nucleotides, and in some embodiments 14 or 15nucleotides, that are necessary to effect specific binding. The onlyapparent limitations on the binding specificity of thetarget/oligonucleotide couples of the presently disclosed subject matterconcern sufficient sequence to be distinctive in the bindingoligonucleotide and sufficient binding capacity of the target substanceto obtain the necessary interaction. Aptamers of binding regionscontaining sequences shorter than 10, e.g., 6-mers, are feasible if theappropriate interaction can be obtained in the context of theenvironment in which the target is placed. Thus, if there is littleinterference by other materials, less specificity and less strength ofbinding can be required.

Aptamers and how to isolate aptamers that bind to specific targets aredisclosed in U.S. Patent Application Publication No. 20030175703 andU.S. Pat. Nos. 5,270,163; 5,567,588; 5,683,867; 6,706,482; 6,855,496;and 7,176,295, the disclosure of each of which is hereby incorporated byreference in its entirety.

IV.D. RNAi-Based Bmx Modulators

In some embodiments, the presently disclosed subject matter takesadvantage of the ability of short, double stranded RNA molecules tocause the down regulation of cellular genes, a process referred to asRNA interference (RNAi). As used herein, “RNA interference” and “RNAi”refer to a process of sequence-specific post-transcriptional genesilencing mediated by a small interfering RNA (siRNA). See generallyFire et al., 1998; U.S. Pat. No. 6,506,559. The process ofpost-transcriptional gene silencing is thought to be an evolutionarilyconserved cellular defense mechanism that has evolved to prevent theexpression of foreign genes (Fire, 1999).

The presence of dsRNA in cells triggers various responses, one of whichis RNAi. RNAi appears to be different from the interferon response todsRNA, which results from dsRNA-mediated activation of an RNA-dependentprotein kinase (PKR) and 2′,5′-oligoadenylate synthetase, resulting innon-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of theenzyme Dicer, a ribonuclease III. Dicer catalyzes the degradation ofdsRNA into short stretches of dsRNA referred to as small interferingRNAs (siRNA; Zamore et al., 2000). The small interfering RNAs thatresult from Dicer-mediated degradation are typically about 21-23nucleotides in length and contain about 19 base pair duplexes. Afterdegradation, the siRNA is incorporated into an endonuclease complexreferred to as an RNA-induced silencing complex (RISC). The RISC iscapable of mediating cleavage of single stranded RNA present within thecell that is complementary to the antisense strand of the siRNA duplex.According to Elbashir et al., cleavage of the target RNA occurs near themiddle of the region of the single stranded RNA that is complementary tothe antisense strand of the siRNA duplex (Elbashir et al., 2001b).

RNAi has been described in several cell type and organisms. Fire et al.,1998 described RNAi in C. elegans. Wianny & Zernicka-Goetz, 1999disclose RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000were able to induce RNAi in Drosophila cells by transfecting dsRNA intothese cells. Elbashir et al. 2001a discloses the presence of RNAi incultured mammalian cells including human embryonic kidney and HeLa cellsby the introduction of duplexes of synthetic 21 nucleotide RNAs.

Experiments using Drosophila embryonic lysates revealed certain aspectsof siRNA length, structure, chemical composition, and sequence that areinvolved in RNAi activity. See Elbashir et al., 2001c. In the disclosedassay, 21 nucleotide siRNA duplexes were most active when they contain3′-overhangs of two nucleotides. Also, the position of the cleavage sitein the target RNA was shown to be defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001b).

Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of a siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5-phosphate moiety onthe siRNA (Nykanen et al., 2001). Other modifications that might betolerated when introduced into an siRNA molecule include modificationsof the sugar-phosphate backbone or the substitution of the nucleosidewith at least one of a nitrogen or sulfur heteroatom (PCT InternationalPublication Nos. WO 00/44914 and WO 01/68836) and certain nucleotidemodifications that might inhibit the activation of double strandedRNA-dependent protein kinase (PKR), specifically 2′-amino or 2′-O-methylnucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge(Canadian Patent Application No. 2,359,180).

Other references disclosing the use of dsRNA and RNAi include PCTInternational Publication Nos. WO 01/75164 (in vitro RNAi system usingcells from Drosophila and the use of specific siRNA molecules forcertain functional genomic and certain therapeutic applications); WO01/36646 (methods for inhibiting the expression of particular genes inmammalian cells using dsRNA molecules); WO 99/32619 (methods forintroducing dsRNA molecules into cells for use in inhibiting geneexpression); WO 01/92513 (methods for mediating gene suppression byusing factors that enhance RNAi); WO 02/44321 (synthetic siRNAconstructs); WO 00/63364 and WO 01/04313 (methods and compositions forinhibiting the function of polynucleotide sequences); and WO 02/055692and WO 02/055693 (methods for inhibiting gene expression using RNAi).

In some embodiments, the modulator of a biological activity of a Bmxgene product comprises an siRNA construct targeted to or against a Bmxgene product (e.g., a subsequence of an RNA molecule transcribed from aBmx gene).

As used herein, the phrase “target RNA” refers to an RNA molecule (forexample, an mRNA molecule encoding a Bmx gene product) that is a targetfor downregulation. Similarly, the phrase “target site” refers to asequence within a target RNA that is “targeted” for cleavage mediated byan siRNA construct that contains sequences within its antisense strandthat are complementary to the target site. Also similarly, the phrase“target cell” refers to a cell that expresses a target RNA and intowhich an siRNA is intended to be introduced. A target cell is in someembodiments a cell in a subject. For example, a target cell can comprisea tumor cell and/or a cell in tumor vasculature that expresses a Bmxgene. Non-limiting examples of sequences encoding target RNA moleculesof the presently disclosed subject matter are presented in Table 1.

As used herein, the phrase “detectable level of cleavage” refers to adegree of cleavage of target RNA (and formation of cleaved product RNAs)that is sufficient to allow detection of cleavage products above thebackground of RNAs produced by random degradation of the target RNA.Production of siRNA-mediated cleavage products from at least 1-5% of thetarget RNA is sufficient to allow detection above background for mostdetection methods.

The terms “small interfering RNA”, “short interfering RNA”, and “siRNA”are used interchangeably and refer to any nucleic acid molecule capableof mediating RNA interference (RNAi) or gene silencing. See e.g., Bass,2001; Elbashir et al., 2001a; and PCT International Publication Nos. WO00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO99/07409, and WO 00/44914. In some embodiments, the siRNA comprises adouble stranded polynucleotide molecule comprising complementary senseand antisense regions, wherein the antisense region comprises a sequencecomplementary to a region of a target nucleic acid molecule (forexample, an mRNA encoding a Bmx polypeptide). In some embodiments, thesiRNA comprises a single stranded polynucleotide havingself-complementary sense and antisense regions, wherein the antisenseregion comprises a sequence complementary to a region of a targetnucleic acid molecule. In some embodiments, the siRNA comprises a singlestranded polynucleotide having one or more loop structures and a stemcomprising self complementary sense and antisense regions, wherein theantisense region comprises a sequence complementary to a region of atarget nucleic acid molecule, and wherein the polynucleotide can beprocessed either in vivo or in vitro to generate an active siRNA capableof mediating RNAi. An siRNA that forms such a stem-and-loop structure(i.e., a “hairpin”) is also referred to herein as a “short hairpin RNA(shRNA)”. As used herein, siRNA molecules need not be limited to thosemolecules containing only RNA, but further encompass chemically modifiednucleotides and non-nucleotides.

The siRNA molecules of the presently disclosed subject matter include,but are not limited to an siRNA molecule of the general structuredepicted in FIG. 7. For the double-stranded molecule shown in FIG. 7, Ncan be any nucleotide, provided that in the loop structure identified asN₅₋₉, all 5-9 nucleotides remain in a single-stranded conformation.Similarly, N₂₋₈ can be any sequence of 2-8 nucleotides or modifiednucleotides, provided that the nucleotides remain in a single-strandedconformation in the siRNA molecule. The duplex represented in FIG. 7 as“19-30 bases of Bmx” can be formed using any contiguous 19-30 basesequence of one of the Bmx gene products disclosed herein (for example,in Table 1). In constructing an siRNA molecule of the presentlydisclosed subject matter, this 19-30 base sequence is followed (in a 5′to 3′ direction) by 5-9 random nucleotides (N₅₋₉ above), thereverse-complement of the 19-30 base sequence, and finally 2-8 randomnucleotides (N₂₋₈ above).

The term “gene expression” generally refers to the cellular processes bywhich a biologically active polypeptide is produced from a DNA sequenceand exhibits a biological activity in a cell. As such, gene expressioninvolves the processes of transcription and translation, but alsoinvolves post-transcriptional and post-translational processes that caninfluence a biological activity of a gene or gene product. Theseprocesses include, but are not limited to RNA syntheses, processing, andtransport, as well as polypeptide synthesis, transport, andpost-translational modification of polypeptides. Additionally, processesthat affect protein-protein interactions within the cell can also affectgene expression as defined herein.

As used herein, the term “modulate” can refer to a change in theexpression level of a gene, or a level of RNA molecule or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits is up regulated or downregulated, such that expression, level, or activity is greater than orless than that observed in the absence of the modulator. For example,the term “modulate” can mean “inhibit” or “suppress”, but the use of theword “modulate” is not limited to this definition.

As used herein, the terms “inhibit”, “suppress”, “down regulate”, andgrammatical variants thereof are used interchangeably and refer to anactivity whereby gene expression or a level of an RNA encoding one ormore gene products is reduced below that observed in the absence of anucleic acid molecule of the presently disclosed subject matter. In someembodiments, inhibition with an siRNA molecule results in a decrease inthe steady state level of a target RNA. In some embodiments, inhibitionwith a siRNA molecule results in an expression level of a target genethat is below that level observed in the presence of an inactive orattenuated molecule that is unable to mediate an RNAi response. In someembodiments, inhibition of gene expression with an siRNA molecule of thepresently disclosed subject matter is greater in the presence of thesiRNA molecule than in its absence. In some embodiments, inhibition ofgene expression is associated with an enhanced rate of degradation ofthe mRNA encoded by the gene (for example, by RNAi mediated by ansiRNA).

As used herein, the terms “gene” and “target gene” refer to a nucleicacid that encodes an RNA such as, but not limited to a nucleic acidsequence that encodes a Bmx polypeptide. The term “gene” also refersbroadly to any segment of DNA associated with a biological function. Assuch, the term “gene” encompasses sequences including but not limited toa coding sequence, a promoter region, a transcriptional regulatorysequence, a non-expressed DNA segment that is a specific recognitionsequence for regulatory proteins, a non-expressed DNA segment thatcontributes to gene expression, a DNA segment designed to have desiredparameters, or combinations thereof. A gene can be obtained by a varietyof methods, including cloning from a biological sample, synthesis basedon known or predicted sequence information, and recombinant derivationof an existing sequence. In some embodiments, a gene is a Bmx gene.Representative Bmx genes correspond to the sequences set forth in Table1, although this list is not intended to be exhaustive.

As is understood in the art, a gene can comprise a coding strand and anon-coding strand. As used herein, the terms “coding strand” and “sensestrand” are used interchangeably, and refer to a nucleic acid sequencethat has the same sequence of nucleotides as an mRNA from which the geneproduct is translated. As is also understood in the art, when the codingstrand and/or sense strand is used to refer to a DNA molecule, thecoding/sense strand includes thymidine residues instead of the uridineresidues found in the corresponding mRNA. Additionally, when used torefer to a DNA molecule, the coding/sense strand can also includeadditional elements not found in the mRNA including, but not limited topromoters, enhancers, and introns. Similarly, the terms “templatestrand” and “antisense strand” are used interchangeably and refer to anucleic acid sequence that is complementary to the coding/sense strand.

The term “complementary sequences”, as used herein, indicates twonucleotide sequences that comprise antiparallel nucleotide sequencescapable of pairing with one another upon formation of hydrogen bondsbetween base pairs. As used herein, the term “complementary sequences”means nucleotide sequences which are substantially complementary, as canbe assessed by the same nucleotide comparison set forth above, or isdefined as being capable of hybridizing to the nucleic acid segment inquestion under relatively stringent conditions such as those describedherein.

Thus, the terms “complementarity” and “complementary” refer to a nucleicacid that can form one or more hydrogen bonds with another nucleic acidsequence by either traditional Watson-Crick or other non-traditionaltypes of interactions. In reference to the nucleic molecules of thepresently disclosed subject matter, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed (e.g., RNAiactivity. For example, the degree of complementarity between the senseand antisense strands of the siRNA construct can be the same ordifferent from the degree of complementarity between the antisensestrand of the siRNA and the target nucleic acid sequence.Complementarity to the target sequence of less than 100% in theantisense strand of the siRNA duplex, including point mutations, is notwell tolerated when these changes are located between the 3′-end and themiddle of the antisense siRNA, whereas mutations near the 5′-end of theantisense siRNA strand can exhibit a small degree of RNAi activity(Elbashir et al., 2001c). Determination of binding free energies fornucleic acid molecules is well known in the art. See e.g., Freier etal., 1986; Turner et al., 1987.

As used herein, the phrase “percent complementarity” refers to thepercentage of contiguous residues in a nucleic acid molecule that canform hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%,70%, 80%, 90%, and 100% complementary). The terms “100% complementary”,“fully complementary”, and “perfectly complementary” indicate that allof the contiguous residues of a nucleic acid sequence can hydrogen bondwith the same number of contiguous residues in a second nucleic acidsequence.

The siRNA molecules of the presently disclosed subject matter can beadded directly to a cell, or can be complexed with cationic lipids,packaged within liposomes, or otherwise delivered to target cells ortissues. The nucleic acid or nucleic acid complexes can be locallyadministered to relevant tissues ex vivo, or in vivo through injection,infusion pump or stent, with or without their incorporation intobiopolymers. The siRNA molecule of the presently disclosed subjectmatter can be encoded by a recombinant vector (for example, a viralvector).

As used herein, the term “RNA” refers to a molecule comprising at leastone ribonucleotide residue. By “ribonucleotide” is meant a nucleotidewith a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety.The terms encompass double stranded RNA, single stranded RNA, RNAs withboth double stranded and single stranded regions, isolated RNA such aspartially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA, or analog RNA, thatdiffers from naturally occurring RNA by the addition, deletion,substitution, and/or alteration of one or more nucleotides. Suchalterations can include addition of non-nucleotide material, such as tothe end(s) of the siRNA or internally, for example at one or morenucleotides of the RNA. Nucleotides in the RNA molecules of thepresently disclosed subject matter can also comprise non-standardnucleotides, such as non-naturally occurring nucleotides or chemicallysynthesized nucleotides or deoxynucleotides. These altered RNAs can bereferred to as analogs or analogs of a naturally occurring RNA.

As used herein, the phrase “double stranded RNA” refers to an RNAmolecule at least a part of which is in Watson-Crick base pairingforming a duplex. As such, the term is to be understood to encompass anRNA molecule that is either fully or only partially double stranded.Exemplary double stranded RNAs include, but are not limited to moleculescomprising at least two distinct RNA strands that are either partiallyor fully duplexed by intermolecular hybridization. Additionally, theterm is intended to include a single RNA molecule that by intramolecularhybridization can form a double stranded region (for example, ahairpin). Thus, as used herein the phrases “intermolecularhybridization” and “intramolecular hybridization” refer to doublestranded molecules for which the nucleotides involved in the duplexformation are present on different molecules or the same molecule,respectively.

As used herein, the phrase “double stranded region” refers to any regionof a nucleic acid molecule that is in a double stranded conformation viahydrogen bonding between the nucleotides including, but not limited tohydrogen bonding between cytosine and guanosine, adenosine andthymidine, adenosine and uracil, and any other nucleic acid duplex aswould be understood by one of ordinary skill in the art. The length ofthe double stranded region can vary from about 15 consecutive basepairsto several thousand basepairs. In some embodiments, the double strandedregion is at least 15 basepairs, in some embodiments between 15 and 50basepairs, and in some embodiments between 15 and 30 basepairs. In someembodiments, the length of the double stranded region is selected fromthe group consisting of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and30 basepairs. In some embodiments, the double stranded region comprisesa first strand comprising a ribonucleotide sequence that corresponds toa coding strand of the Bmx gene and a second strand comprising aribonucleotide sequence that is complementary to the first strand, andwherein the first strand and the second strand hybridize to each otherto form the double-stranded molecule. As used herein, the terms“corresponds to”, “corresponding to”, and grammatical variants thereofrefer to a nucleotide sequence that is 100% identical to at least 19contiguous nucleotides of a nucleic acid sequence of a Bmx gene. Thus, afirst nucleic acid sequence that “corresponds to” a coding strand of aBmx gene is a nucleic acid sequence that is 100% identical to at least19 contiguous nucleotides of a Bmx gene, including, but note limited to5′ untranslated sequences, exon sequences, intron sequences, and 3′untranslated sequences.

In a representative embodiment, the length of the double stranded regionis 19 basepairs. As describe hereinabove, the formation of the doublestranded region results from the hybridization of complementary RNAstrands (for example, a sense strand and an antisense strand), eithervia an intermolecular hybridization (i.e., involving 2 or more distinctRNA molecules) or via an intramolecular hybridization, the latter ofwhich can occur when a single RNA molecule contains self-complementaryregions that are capable of hybridizing to each other on the same RNAmolecule. These self-complementary regions are typically separated by ashort stretch of nucleotides (for example, about 5-10 nucleotides) suchthat the intramolecular hybridization event forms what is referred to inthe art as a “hairpin”.

The nucleic acid molecules of the presently disclosed subject matterindividually, or in combination or in conjunction with other drugs, canbe used to treat diseases or conditions discussed herein. For example,to treat a particular disease or condition, the siRNA molecules can beadministered to a subject or can be administered to other appropriatecells evident to those skilled in the art, individually or incombination with one or more drugs under conditions suitable for thetreatment.

An exemplary nucleotide sequence employed in the methods disclosedherein comprises sequences that are complementary to each other, thecomplementary regions being capable of forming a duplex of in someembodiments at least about 15 to 50 basepairs. One strand of the duplexcomprises a nucleic acid sequence of at least 15 contiguous bases havinga nucleic acid sequence of a nucleic acid molecule of the presentlydisclosed subject matter (for example, those nucleic acid sequences thatcorrespond to the GENBANK® Accession Nos. set forth in Table 1). In someembodiments, one strand of the duplex comprises a nucleic acid sequencecomprising 15 to 18 nucleotides, or even longer where desired, such as19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides or up tothe full length of any of those nucleic acid sequences that correspondto the GENBANK® Accession Nos. set forth in Table 1, or any other Bmxtranscription product. Such fragments can be readily prepared by, forexample, directly synthesizing the fragment by chemical synthesis, byapplication of nucleic acid amplification technology, or by introducingselected sequences into recombinant vectors for recombinant production.The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex nucleic acid mixture (e.g., total cellular DNA or RNA).

The term “elongated sequence” refers to an addition of nucleotides (orother analogous molecules) incorporated into the nucleic acid. Forexample, a polymerase (e.g., a DNA polymerase) can add sequences at the3′ terminus of the nucleic acid molecule. In addition, the nucleotidesequence can be combined with other DNA sequences, such as promoters,promoter regions, enhancers, polyadenylation signals, intronicsequences, additional restriction enzyme sites, multiple cloning sites,and other coding segments.

The phrases “operatively linked” and “operably linked”, as used herein,refer to a functional combination between a promoter region and anucleotide sequence such that the transcription of the nucleotidesequence is controlled and regulated by the promoter region. Similarly,a nucleotide sequence is said to be under the “transcriptional control”of a promoter to which it is operably linked. Techniques for operativelylinking a promoter region to a nucleotide sequence are known in the art.The phrases are also intended to refer to functional combinationsbetween promoter regions and/or nucleotide sequences and/or othernucleotide sequence features that interact with the promoter regionsand/or the nucleotide sequences to affect transcription of thenucleotide sequences and/or the nature of the transcript produced.Exemplary nucleotide sequence features that can also be operably linkedto promoters and/or nucleotide sequences include, but are not limited toenhancers, suppressors, transcription termination signals,polyadenylation signals, etc.

The term “heterologous”, as used herein to refer to a promoter or anyother nucleic acid, refers to a sequence that originates from a sourceforeign to an intended host cell or, if from the same source, ismodified from its original form. Thus, a heterologous nucleic acid in ahost cell includes a gene that is endogenous to the particular host cellbut has been modified, for example by mutagenesis or by isolation fromnative cis-regulatory sequences.

The terms “heterologous gene”, “heterologous DNA sequence”,“heterologous nucleotide sequence”, “exogenous nucleic acid molecule”,or “exogenous DNA segment”, as used herein, each refer to a sequencethat originates from a source foreign to an intended host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified, for example bymutagenesis or by isolation from native transcriptional regulatorysequences. The terms also include non-naturally occurring multiplecopies of a naturally occurring nucleotide sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid wherein the element is not ordinarily found.

The term “recombinant” generally refers to an isolated nucleic acid thatis replicable in a non-native environment. Thus, a recombinant nucleicacid can comprise a non-replicable nucleic acid in combination withadditional nucleic acids, for example vector nucleic acids, which enableits replication in a host cell.

The term “vector” is used herein to refer to a nucleic acid moleculehaving nucleotide sequences that enable its replication in a host cell.A vector can also include nucleotide sequences to permit ligation ofnucleotide sequences within the vector, wherein such nucleotidesequences are also replicated in a host cell. Representative vectorsinclude plasmids, cosmids, and viral vectors. A vector can also mediaterecombinant production of a soluble peptide or polypeptide of thepresently disclosed subject matter.

The terms “expression vector” and “expression construct” as used hereinrefer to a DNA sequence capable of directing expression of a particularnucleotide sequence in an appropriate host cell, comprising a promoteroperatively linked to the nucleotide sequence of interest which isoperably linked to termination signals. It also typically comprisessequences required for proper translation of the nucleotide sequence.The construct comprising the nucleotide sequence of interest can bechimeric. The construct can also be one that is naturally occurring buthas been obtained in a recombinant form useful for heterologousexpression.

The term “construct”, as used herein to describe an expressionconstruct, refers to a vector further comprising a nucleotide sequenceoperably inserted with the vector, such that the nucleotide sequence isexpressed.

The terms “recombinantly expressed” or “recombinantly produced” are usedinterchangeably to generally refer to the process by which a polypeptideencoded by a recombinant nucleic acid is produced.

The term “heterologous expression system” refers to a host cellcomprising a heterologous nucleic acid and the polypeptide encoded bythe heterologous nucleic acid. For example, a heterologous expressionsystem can comprise a host cell transfected with a construct comprisinga recombinant nucleic acid, or a cell line produced by introduction ofheterologous nucleic acids into a host cell genome.

The term “promoter” or “promoter region” each refers to a nucleotidesequence within a gene that is positioned 5′ to a coding sequence of asame gene and functions to direct transcription of the coding sequence.The promoter region comprises a transcriptional start site, and canadditionally include one or more transcriptional regulatory elements. Insome embodiments, a method of the presently disclosed subject matteremploys a hypoxia inducible promoter.

A “minimal promoter” is a nucleotide sequence that has the minimalelements required to enable basal level transcription to occur. As such,minimal promoters are not complete promoters but rather are subsequencesof promoters that are capable of directing a basal level oftranscription of a reporter construct in an experimental system. Minimalpromoters include but are not limited to the CMV minimal promoter, theHSV-tk minimal promoter, the simian virus 40 (SV40) minimal promoter,the human β-actin minimal promoter, the human EF2 minimal promoter, theadenovirus E1B minimal promoter, and the heat shock protein (hsp) 70minimal promoter. Minimal promoters are often augmented with one or moretranscriptional regulatory elements to influence the transcription of anoperably linked gene. For example, cell-type-specific or tissue-specifictranscriptional regulatory elements can be added to minimal promoters tocreate recombinant promoters that direct transcription of an operablylinked nucleotide sequence in a cell-type-specific or tissue-specificmanner

Different promoters have different combinations of transcriptionalregulatory elements. Whether or not a gene is expressed in a cell isdependent on a combination of the particular transcriptional regulatoryelements that make up the gene's promoter and the differenttranscription factors that are present within the nucleus of the cell.As such, promoters are often classified as “constitutive”,“tissue-specific”, “cell-type-specific”, or “inducible”, depending ontheir functional activities in vivo or in vitro. For example, aconstitutive promoter is one that is capable of directing transcriptionof a gene in a variety of cell types. Exemplary constitutive promotersinclude the promoters for the following genes which encode certainconstitutive or “housekeeping” functions: hypoxanthine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR; (Scharfmann et al.,1991), adenosine deaminase, phosphoglycerate kinase (PGK), pyruvatekinase, phosphoglycerate mutase, the β-actin promoter (see e.g.,Williams et al., 1993), and other constitutive promoters known to thoseof skill in the art. “Tissue-specific” or “cell-type-specific”promoters, on the other hand, direct transcription in some tissues andcell types but are inactive in others. Exemplary tissue-specificpromoters include the PSA promoter (Yu et al., 1999; Lee et al., 2000),the probasin promoter (Greenberg et al., 1994; Yu et al., 1999), and theMUC1 promoter (Kurihara et al., 2000) as discussed above, as well asother tissue-specific and cell-type specific promoters known to those ofskill in the art.

When used in the context of a promoter, the term “linked” as used hereinrefers to a physical proximity of promoter elements such that theyfunction together to direct transcription of an operably linkednucleotide sequence

The term “transcriptional regulatory sequence” or “transcriptionalregulatory element”, as used herein, each refers to a nucleotidesequence within the promoter region that enables responsiveness to aregulatory transcription factor. Responsiveness can encompass a decreaseor an increase in transcriptional output and is mediated by binding ofthe transcription factor to the DNA molecule comprising thetranscriptional regulatory element.

The term “transcription factor” generally refers to a protein thatmodulates gene expression by interaction with the transcriptionalregulatory element and cellular components for transcription, includingRNA Polymerase, Transcription Associated Factors (TAFs),chromatin-remodeling proteins, and any other relevant protein thatimpacts gene transcription.

In some embodiments, a promoter that is operably linked to a nucleotidesequence encoding a modulator of Bmx is a promoter that is expressed ina cell that expresses Bmx. An exemplary promoter would be a Bmx promoteritself, in some embodiments the promoter of the Bmx gene from the samespecies in which the compositions and methods of the presently disclosedsubject matter are to be deployed. For example, human Bmx gene productscorrespond to GENBANK® Accession Nos. NM_(—)203281 (SEQ ID NO: 1) andNM_(—)001721 (SEQ ID NO: 3). These sequences are present on humanchromosome X, and the genomic sequence that corresponds to the firstnucleotide of NM_(—)203281 is found on the plus strand GENBANK®Accession No. NT_(—)011757.15 at position 13,300,583. One of ordinaryskill in the art could thus, with routine experimentation, isolate afragment of human chromosome X in the vicinity of position 13,300,583 ofGENBANK® Accession No. NT_(—)011757.15 that corresponds to the Bmxpromoter. Once isolated, the promoter fragment can be operably linked toa nucleotide sequence encoding a modulator of Bmx, thereby increasingthe likelihood that the modulator of Bmx and Bmx would be co-expressedin a cell type of interest.

Alternatively or in addition, a promoter that includes one or morehypoxia response elements (HREs) can be operably linked to a nucleotidesequence encoding a modulator of Bmx in order to express the modulatorof Bmx in hypoxic cells (e.g., in hypoxic regions of a tumor). Arepresentative promoter that contains HRE sequences is the vascularendothelial growth factor (VEGF) promoter. Other HRE-containingpromoters are disclosed in U.S. Pat. No. 7,067,649. Additionally,Semenza & Wang, 1992; Blanchard et al., 1992; Firth et al., 1995; andEbert & Bunn, 1998 teach sequences of HREs, which can be concatamerizedand included with one or more minimal promoter elements to produce asynthetic hypoxia-responsive promoter.

The presently disclosed subject matter includes in some embodimentsvectors encoding Bmx modulators (e.g., siRNAs targeted to Bmx,antibodies or fragments or derivatives thereof that bind to Bmx, etc.).The term “vector”, as used herein refers to a DNA molecule havingsequences that enable the transfer of those sequences to a compatiblehost cell. A vector also includes nucleotide sequences to permitligation of nucleotide sequences within the vector, wherein suchnucleotide sequences are also replicated in a compatible host cell. Avector can also mediate recombinant production of a therapeuticpolypeptide, as described further herein below. In some embodiments, avector is an adenovirus vector or an adeno-associated virus vector.

Nucleic acids of the presently disclosed subject matter can be cloned,synthesized, recombinantly altered, mutagenized, or combinationsthereof. Standard recombinant DNA and molecular cloning techniques usedto isolate nucleic acids are known in the art. Exemplary, non-limitingmethods are described by Silhavy et al., 1984; Ausubel et al., 1992;Ausubel, 1995; Glover & Hames, 1995; and Sambrook & Russell, 2001).Site-specific mutagenesis to create base pair changes, deletions, orsmall insertions is also known in the art as exemplified by publications(see e.g., Adelman et al., 1983; Sambrook & Russell, 2001).

In some embodiments, the presently disclosed subject matter provides ansiRNA molecule that has been synthesized outside of a target cell priorto introduction of the siRNA into the target cell. In this embodiment,the synthesis can be performed either mechanically (i.e., using an RNAsynthesis machine) or using recombinant techniques.

Mechanical synthesis of nucleic acids greater than 100 nucleotides inlength is difficult using automated methods, and the cost of suchmolecules tends to be prohibitive. As used herein, small nucleic acidmotifs (“small” referring to nucleic acid motifs in some embodiments nomore than 100 nucleotides in length, in some embodiments no more than 80nucleotides in length, and in some embodiments no more than 50nucleotides in length; e.g., individual siRNA oligonucleotide sequencesor siRNA sequences synthesized in tandem) can be used for exogenousdelivery. The simple structure of these molecules increases the abilityof the nucleic acid to invade targeted regions of protein and/or RNAstructure. Exemplary molecules of the presently disclosed subject matterare chemically synthesized, and others can similarly be synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art. See e.g., Caruthers et al., 1992; PCTInternational Publication No. WO 99/54459; Wincott et al., 1995; Wincott& Usman, 1997; Brennan et al., 1998; and U.S. Pat. No. 6,001,311, eachof which is incorporated herein by reference. The synthesis ofoligonucleotides makes use of common nucleic acid protecting andcoupling groups, such as dimethoxytrityl at the 5′-end andphosphoramidites at the 3′-end. In a non-limiting example, small-scalesyntheses can be conducted on a Applied Biosystems 3400 DNA Synthesizer(Applied Biosystems Inc., Foster City, Calif., United States of America)using a 0.2 μmol scale protocol with a 2.5 minute coupling step for2′-O-methylated nucleotides and a 45 second coupling step for 2′-deoxynucleotides or 2′-deoxy-2′-fluoro nucleotides. Alternatively, synthesesat the 0.2 μmol scale can be performed on a 96-well plate synthesizer. A33-fold excess (60 μL of 0.11 M; 6.6 μmol) of 2′-O-methylphosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of0.25 M; 15 μmol) can be used in each coupling cycle of 2′-O-methylresidues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μLof 0.11 M; 4.4 μmol) of deoxy phosphoramidite and a 70-fold excess ofS-ethyl tetrazole (40 μL of 0.25 M; 10 μmol) can be used in eachcoupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the Applied Biosystems 3400 DNA Synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for theApplied Biosystems 3400 DNA Synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (AppliedBiosystems, Inc.); capping is performed with 16% N-methyl imidazole inTHF (Applied Biosystems, Inc.) and 10% acetic anhydride/10% 2,6-lutidinein THF (Applied Biosystems, Inc.); and oxidation solution is 16.9 mM I₂,49 mM pyridine, 9% water in tetrahydrofuran (THF; PerSeptive Biosystmes,Hamburg, Germany). Synthesis Grade acetonitrile (Honeywell BURDICK &JACKSON™, Morristown, N.J., United States of America) is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate internucleotide linkages, Beaucage reagent(³H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H₂O (3:1:1), vortexed, andthe supernatant is added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

In some embodiments, the method of synthesis used for RNA includingcertain siRNA molecules of the presently disclosed subject matterfollows the procedure as described in Usman et al., 1987; Scaringe etal., 1990; Wincott et al., 1995; Wincott & Usman, 1997; and makes use ofcommon nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small-scale syntheses are conducted on an AppliedBiosystems 3400 DNA Synthesizer using a 0.2 μmol scale protocol with a7.5 minute coupling step for alkylsilyl protected nucleotides and a 2.5minute coupling step for 2′-O-methylated nucleotides. Alternatively,syntheses at the 0.2 μmol scale can be done on a 96-well platesynthesizer. A 33-fold excess (60 μL of 0.11 M; 6.6 μmol) of 2′-O-methylphosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25M; 15 μmol) can be used in each coupling cycle of 2′-O-methyl residuesrelative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11M; l 13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M; 30 μmol) can beused in each coupling cycle of ribo residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the Applied Biosystems 3400 DNASynthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the Applied Biosystems 3400 DNA Synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride(Applied Biosystems, Inc.); capping is performed with 16% N-methylimidazole in THF (Applied Biosystems, Inc.) and 10% acetic anhydride/10%2,6-lutidine in THF (Applied Biosystems, Inc.); oxidation solution is16.9 mM I₂, 49 mM pyridine, 9% water in tetrahydrofuran (THF; PerSeptiveBiosystmes, Hamburg, Germany). Synthesis Grade acetonitrile (HoneywellBURDICK & JACKSON™, Morritown, N.J., United States of America) is useddirectly from the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. (Natick, Mass., United States of America).Alternately, for the introduction of phosphorothioate linkages, Beaucagereagent (³H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile)is used.

Deprotection of the RNA can be performed, for example, using either atwo-pot or one-pot protocol. For the two-pot protocol, the polymer-boundtrityl-on oligoribonucleotide is transferred to a 4 mL glass screw topvial and suspended in a solution of 40% aqueous methylamine (1 mL) at65° C. for 10 minutes. After cooling to −20° C., the supernatant isremoved from the polymer support. The support is washed three times with1.0 mL of EtOH:MeCN:H2O (3:1:1), vortexed, and the supernatant is thenadded to the first supernatant. The combined supernatants, containingthe oligoribonucleotide, are dried to a white powder. The basedeprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMPsolution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μLTEA and 1 mL TEA.3HF to provide a 1.4 M HF concentration) and heated to65° C. After 1.5 hours, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine:DMSO (1:1; 0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperature,TEA.3HF (0.1 mL) is added, and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C., and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5%trifluoroacetic acid (TFA) for 13 min. The cartridge is then washedagain with water, salt exchanged with 1 M NaCl, and washed with wateragain. The oligonucleotide is then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically greater than 98%(Wincott et al., 1995). Those of ordinary skill in the art willrecognize that the scale of synthesis can be adapted to be larger orsmaller than the example described above including but not limited to96-well format: all that is important is the ratio of chemicals used inthe reaction.

Alternatively, the nucleic acid molecules of the presently disclosedsubject matter can be synthesized separately and joined togetherpost-synthetically, for example, by ligation (PCT InternationalPublication No. WO 93/23569; Shabarova et al., 1991; Bellon et al.,1997), or by hybridization following synthesis and/or deprotection.

The siRNA molecules of the presently disclosed subject matter can alsobe synthesized via a tandem synthesis methodology, wherein both siRNAstrands are synthesized as a single contiguous oligonucleotide fragmentor a strand separated by a linker which, in some embodiments, issubsequently cleaved to provide separate siRNA fragments or strands thathybridize and permit purification of the siRNA duplex. The linker can bea polynucleotide linker or a non-nucleotide linker. The tandem synthesisof siRNA can be readily adapted to both multiwell and multiplatesynthesis platforms such as 96 well or similarly larger multi-wellplatforms. The tandem synthesis of siRNA can also be readily adapted tolarge-scale synthesis platforms employing batch reactors, synthesiscolumns and the like.

A siRNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

In some embodiments, recombinant techniques can be used to synthesize ansiRNA, which can thereafter be purified from the source and transferredto a target cell. There are many techniques that are known in the artfor the synthesis RNA molecules in recombinant cells, and any suchtechnique can be used in the practice of the presently disclosed subjectmatter. One such general strategy for synthesizing an RNA moleculeincludes cloning a DNA sequence downstream of an RNA polymerase promoterand introducing the recombinant molecule into a cell in which thepromoter is competent to direct transcription of the cloned sequence.This can be accomplished using a plasmid constructed for this purpose.

Alternatively, the RNA can be synthesized in the target cell using anexpression vector, for example an expression plasmid. Such plasmidsinclude, but are not limited to the pSILENCER™ series of plasmids(Ambion, Inc., Austin, Tex., United States of America), and the plasmiddisclosed by Miyagishi & Taira, 2002.

The pSILENCER™ series of plasmids contain a cloning site downstream of amammalian RNA polymerase III promoter. A nucleic acid encoding a hairpinwith a 19 base pair duplex region can be cloned into the cloning site ofone of these plasmids. When the recombinant plasmid is introduced into amammalian cell, the RNA polymerase III promoter directs transcription ofthe hairpin RNA molecule, which thereafter forms the hairpincharacterized by the 19 base pair duplex. This hairpin is apparentlyrecognized by the Dicer nuclease, which cleaves the hairpin to form afunctional siRNA.

Mivagishi & Taira, 2002 discloses another strategy for producing siRNAmolecules. This reference discloses a plasmid that has two RNApolymerase III promoters. To produce an siRNA, the same 19 base pairnucleic acid molecule is cloned downstream of each promoter, but inopposite orientations. Thus, the plasmid produces distinct sense andantisense RNA strands, which then undergo intermolecular hybridizationto produce an siRNA. In this case, the promoter is the U6 promoter. AnRNA transcribed from a U6 promoter has a stretch of about four uridinesat its 3′ end. Thus, the use of this plasmid results in the productionof two RNA strands, each of which contains a 19 base region that iscapable of hybridizing to a 19 base region in the other, with a short 3′overhang.

Chemically synthesizing nucleic acid molecules incorporating variousmodifications (e.g., to base, sugar, and/or phosphate moieties) canreduce the degradation of the nucleic acid molecules by ribonucleasespresent in biological fluids, and can thus can increase the potency oftherapeutic nucleic acid molecules (see e.g., PCT InternationalPublication Nos. WO 92/07065, WO 93/15187, and WO 91/03162; U.S. Pat.Nos. 5,334,711 and 6,300,074; Perrault et al., 1990; Pieken et al.,1991; Usman & Cedergren, 1992; Limbach et al., 1994; Burgin et al.,1996; Usman et al., 1996; all of which are incorporated by referenceherein). Each of the above references describes various chemicalmodifications that can be made to the base, phosphate, and/or sugarmoieties of the nucleic acid molecules described herein. Modificationscan be employed to enhance the efficacy of the disclosed nucleic acidmolecules in cells. Some of the non-limiting examples of basemodifications that can be introduced into nucleic acid moleculesinclude, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,5-bromouridine), 6-azapyrimidines and 6-alkylpyrimidines (e.g.6-methyluridine), propyne, and others (Burgin et al., 1996; Uhlman &Peyman, 1990).

There are several examples in the art describing sugar, base, andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides can be modified to enhance theirstability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (reviewedin Usman & Cedergren, 1992; Usman et al., 1994; Burgin et al., 1996).Sugar modification of nucleic acid molecules have been extensivelydescribed in the art (see PCT International Publication Nos. WO92/07065, WO 93/15187, WO 98/13526, and WO 97/26270; U.S. Pat. Nos.5,334,711; 5,716,824; and 5,627,053; Perrault et al., 1990; Pieken etal., 1991; Usman & Cedergren, 1992; Beigelman et al., 1995; Karpeisky etal., 1998; Earnshaw & Gait, 1998; Verma & Eckstein, 1998; Burlina etal., 1997; all of which are incorporated by reference herein). Suchpublications describe general methods and strategies to determine thelocation of incorporation of sugar, base, and/or phosphate modificationsand the like into nucleic acid molecules without modulating catalysis.In view of such teachings, similar modifications can be used asdescribed herein to modify the siRNA nucleic acid molecules of thepresently disclosed subject matter so long as the ability of the siRNAsto promote RNAi in a cell is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate and/or 5′-methylphosphonate linkages improvesstability, excessive modifications can cause toxicity or decreasedactivity. Therefore, when designing nucleic acid molecules, the numberof these internucleotide linkages should be minimized. Reducing theconcentration of these linkages should lower toxicity, resulting inincreased efficacy and higher specificity of these molecules.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see e.g., Loakes, 2001).

Small interfering RNA (siRNA) molecules having chemical modificationsthat maintain or enhance activity are provided. Such a nucleic acid isalso generally more resistant to nucleases than an unmodified nucleicacid. Accordingly, the in vitro and/or in vivo activity should not besignificantly lowered. In cases in which modulation is the goal, nucleicacid molecules delivered exogenously should optimally be stable withincells until translation of the target RNA has been modulated long enoughto reduce the levels of the undesirable protein. This period of timevaries between hours to days depending upon the disease state.Improvements in the chemical synthesis of RNA (Wincott et al., 1995;Caruthers et al., 1992) have expanded the ability to modify nucleic acidmolecules by introducing nucleotide modifications to enhance theirnuclease stability, as described above. siRNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., 1995) andre-suspended in water.

In some embodiments, the presently disclosed subject matter featuresconjugates and/or complexes of siRNA molecules. Such conjugates and/orcomplexes can be used to facilitate delivery of siRNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the presently disclosed subject matter can impart therapeuticactivity by transferring therapeutic compounds across cellularmembranes, altering the pharmacokinetics of, and/or modulating thelocalization of nucleic acid molecules of the presently disclosedsubject matter. The presently disclosed subject matter encompasses thedesign and synthesis of novel conjugates and complexes for the deliveryof molecules, including, but not limited to, small molecules, lipids,phospholipids, nucleosides, nucleotides, nucleic acids, antibodies,toxins, negatively charged polymers, and other polymers, for exampleproteins, peptides, hormones, carbohydrates, polyethylene glycols, orpolyamines, across cellular membranes. In general, the transportersdescribed are designed to be used either individually or as part of amulti-component system, with or without degradable linkers. Thesecompounds are expected to improve delivery and/or localization ofnucleic acid molecules of the presently disclosed subject matter into anumber of cell types originating from different tissues, in the presenceor absence of serum (see U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siRNA molecule of the presentlydisclosed subject matter or the sense and antisense strands of a siRNAmolecule of the presently disclosed subject matter. The biodegradablelinker is designed such that its stability can be modulated for aparticular purpose, such as delivery to a particular tissue or celltype. The stability of a nucleic acid-based biodegradable linkermolecule can be modulated by using various chemistries, for examplecombinations of ribonucleotides, deoxyribonucleotides, and chemicallymodified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino,2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or basemodified nucleotides. The biodegradable nucleic acid linker molecule canbe a dimer, trimer, tetramer or longer nucleic acid molecule, forexample, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprisea single nucleotide with a phosphorus-based linkage, for example, aphosphoramidate or phosphodiester linkage. The biodegradable nucleicacid linker molecule can also comprise nucleic acid backbone, nucleicacid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siRNA molecules either alone or in combination with othermolecules provided by the presently disclosed subject matter includetherapeutically active molecules such as antibodies, hormones,antivirals, peptides, proteins, chemotherapeutics, small molecules,vitamins, co-factors, nucleosides, nucleotides, oligonucleotides,enzymatic nucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siRNA, dsRNA, allozymes, aptamers,decoys, and analogs thereof. Biologically active molecules of thepresently disclosed subject matter also include molecules capable ofmodulating the pharmacokinetics and/or pharmacodynamics of otherbiologically active molecules, for example, lipids and polymers such aspolyamines, polyamides, polyethylene glycol, and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Nucleic acid molecules (e.g., siRNA molecules) delivered exogenously areintended to be stable within cells until the level of the target RNA hasbeen reduced sufficiently. The nucleic acid molecules are resistant tonucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the presently disclosed subject matter and in the art haveexpanded the ability to modify nucleic acid molecules by introducingnucleotide modifications to enhance their nuclease stability asdescribed above.

In some embodiments, siRNA molecules having chemical modifications thatmaintain or enhance enzymatic activity of proteins involved in RNAi areprovided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivoactivity should not be significantly lowered.

Use of the nucleic acid-based molecules of the presently disclosedsubject matter will lead to better treatment of the disease progressionby affording the possibility of combination therapies (e.g., multiplesiRNA molecules targeted to different genes; nucleic acid moleculescoupled with known small molecule modulators; or intermittent treatmentwith combinations of molecules, including different motifs and/or otherchemical or biological molecules). The treatment of subjects with siRNAmolecules can also include combinations of different types of nucleicacid molecules, such as enzymatic nucleic acid molecules (ribozymes),allozymes, antisense, 2,5-A oligoadenylate, decoys, aptamers etc.

In another aspect a siRNA molecule of the presently disclosed subjectmatter comprises one or more 5′ and/or 3′-cap structures, for example ononly the sense siRNA strand, antisense siRNA strand, or both siRNAstrands.

As used herein, the phrase “cap structure” is meant to refer to chemicalmodifications that have been incorporated at either terminus of theoligonucleotide (see e.g., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and can help in delivery and/orlocalization within a cell. The cap can be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap), or can be present on bothtermini. In non-limiting examples: the 5′-cap is selected from the groupcomprising inverted abasic residue (moiety); 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety.

In some embodiments, the 3′-cap is selected from a group comprising4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide;4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate;1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexylphosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate;1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modifiedbase nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide;acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide;3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety;5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate;1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridgingor non bridging methylphosphonate and 5′-mercapto moieties (seegenerally Beaucage & Iyer, 1993; incorporated by reference herein).

As used herein, the term “non-nucleotide” refers to any group orcompound which can be incorporated into a nucleic acid chain in theplace of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound is typically abasic, in thatit does not typically contain a commonly recognized nucleotide base,such as adenine (A), guanine (G), cytosine (C), thymine (T), or uracil(U), and therefore lacks a base at the 1′-position.

As used herein, the term “alkyl” group refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain, and cyclic alkylgroups. In some embodiments, the alkyl group has 1 to 12 carbons. Insome embodiments, it is a lower alkyl of from 1 to 7 carbons, and insome embodiments it is a lower alkyl of from 1 to 4 carbons. The alkylgroup can be substituted or unsubstituted. When substituted thesubstituted group(s) is in alternative embodiments, hydroxyl, cyano,alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino, or SH.

The term “alkyl” also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. In someembodiments, the alkenyl group has 1 to 12 carbons. In some embodiments,it is a lower alkenyl of from 1 to 7 carbons, and in some embodiments itis a lower alkenyl of from 1 to 4 carbons. The alkenyl group can besubstituted or unsubstituted. When substituted the substituted group(s)is in alternative embodiments, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂,halogen, N(CH₃)₂, amino, or SH.

The term “alkyl” also includes alkynyl groups that have an unsaturatedhydrocarbon group containing at least one carbon-carbon triple bond,including straight-chain, branched-chain, and cyclic groups. In someembodiments, the alkynyl group has 1 to 12 carbons. In some embodiments,it is a lower alkynyl of from 1 to 7 carbons, and in some embodiments itis a lower alkynyl of from 1 to 4 carbons. The alkynyl group can besubstituted or unsubstituted. When substituted the substituted group(s)is in alternative embodiments, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ orN(CH₃)₂, amino or SH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide, and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl, andbiaryl groups, all of which can be optionally substituted. Exemplarysubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted. An “amide” refers to a —C(O)—NH—R, where R is either alkyl,aryl, alkylaryl, or hydrogen. An “ester” refers to an C(O)—OR′, where Ris either alkyl, aryl, alkylaryl, or hydrogen.

The term “nucleotide” is used herein as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar, and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate, and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides, and other; see e.g., Usman et al., 1996; PCTInternational Publication Nos. WO 92/07065 and WO 93/15187, allincorporated by reference herein). There are several examples ofmodified nucleic acid bases known in the art as summarized by Limbach etal., 1994. Some of the non-limiting examples of base modifications thatcan be introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidinesand 6-alkylpyrimidines (e.g., 6-methyluridine), propyne, and others(Burgin et al., 1996; Uhlman & Peyman, 1990). By “modified bases” inthis aspect is meant nucleotide bases other than adenine, guanine,cytosine, and uracil at 1′ position or their equivalents.

In some embodiments, the presently disclosed subject matter featuresmodified siRNA molecules, with phosphate backbone modificationscomprising one or more phosphorothioate, phosphorodithioate,methylphosphonate, phosphotriester, morpholino, amidate carbamate,carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide,sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.For a review of oligonucleotide backbone modifications, see Hunziker &Leumann, 1995 and De Mesmaeker et al., 1994.

As used herein, the term “abasic” refers to sugar moieties lacking acommonly recognized nucleoside base (e.g., A, C, G, T, or U) or havingother chemical groups in place of the commonly recognized base at the 1′position. See e.g., U.S. Pat. No. 5,998,203.

As used herein, the phrase “unmodified nucleoside” refers to one of thebases adenine, cytosine, guanine, thymine, or uracil joined to the 1′carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate.

In connection with 2′-modified nucleotides as described for thepresently disclosed subject matter, by “amino” is meant 2′—NH₂ or2′-O—NH₂, which can be modified or unmodified. Such modified groups aredescribed, for example, in U.S. Pat. Nos. 5,672,695 and 6,248,878, whichare both incorporated by reference in their entireties.

Various modifications to nucleic acid siRNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and/or ease of introductionof such oligonucleotides to the target site (for example, to enhancepenetration of cellular membranes, and confer the ability to recognizeand bind to targeted cells).

IV.E. Sustained Bioavailability

The term “sustained bioavailability” is used herein to describe acomposition comprising a Bmx antagonist and a carrier, whereby thebioavailability of a Bmx antagonist at a target site is sufficient toachieve radiosensitization of a target (e.g., a tumor). The term“sustained bioavailability” also refers to a bioavailability sufficientto inhibit blood vessel growth of and/or within the target (e.g., atumor and/or the vasculature thereof). The term “sustainedbioavailability” encompasses factors including but not limited tosustained release of a Bmx antagonist from a carrier, metabolicstability of a Bmx antagonist, systemic transport of a compositioncomprising a Bmx antagonist, and effective dose of a Bmx antagonist.

As disclosed herein, an immediate response of tumor blood vessels toradiation is a decrease in tumor blood flow. This response can diminishadministration of an anti-tumor composition (e.g., a Bmx antagonist).Recognizing this response, the disclosure of the presently claimedsubject matter provides that sustained bioavailability of a Bmxantagonist, for example by selection of a carrier and administrationregimen that achieve sustained bioavailability, can improve anti-tumoractivity. One example of carrier comprises a gene therapy vectorencoding a Bmx antagonist (e.g., a neutralizing antibody or fragment orderivative thereof or an siRNA targeted to a Bmx gene product).

A method comprising a carrier or administration approach for sustainedbioavailability can also improve therapies directed toward modulation ofother components of the Bmx signaling pathway. Thus, the presentlyclaimed subject matter provides in some embodiments an improved methodfor inhibiting tumor growth, the method comprising administration of agene therapy vector encoding an inhibitor of Bmx signaling, wherebybioavailability of the inhibitor at a tumor is sustained, and wherebytumor growth delay is improved.

V. Compositions

In accordance with the methods of the presently claimed subject matter,a composition that is administered to increase the radiosensitivity of atarget tissue in a subject comprises: (a) a Bmx antagonist; and (b) apharmaceutically acceptable carrier. Any suitable carrier thatfacilitates drug preparation and/or administration can be used.

V.A. Carriers

The carrier can be a viral vector or a non-viral vector. Suitable viralvectors include adenoviruses, adeno-associated viruses (AAVs),retroviruses, pseudotyped retroviruses, herpes viruses, vacciniaviruses, Semiliki forest virus, and baculoviruses. In some embodimentsof the presently claimed subject matter, the carrier comprises anadenoviral gene therapy construct that encodes a Bmx antagonist.

Suitable non-viral vectors that can be used to deliver a Bmx antagonistinclude but are not limited to a plasmid, a nanosphere (Manome et al.,1994; Saltzman & Fung, 1997), a peptide (U.S. Pat. Nos. 6,127,339 and5,574,172), a glycosaminoglycan (U.S. Pat. No. 6,106,866), a fatty acid(U.S. Pat. No. 5,994,392), a fatty emulsion (U.S. Pat. No. 5,651,991), alipid or lipid derivative (U.S. Pat. No. 5,786,387), collagen (U.S. Pat.No. 5,922,356), a polysaccharide or derivative thereof (U.S. Pat. No.5,688,931), a nanosuspension (U.S. Pat. No. 5,858,410), a polymericmicelle or conjugate (Goldman et al., 1997) and U.S. Pat. Nos.4,551,482, 5,714,166, 5,510,103, 5,490,840, and 5,855,900), and apolysome (U.S. Pat. No. 5,922,545).

Where appropriate, two or more types of carriers can be used together.For example, a plasmid vector can be used in conjunction with liposomes.Currently, some embodiments of the presently claimed subject matterenvisions the use of an adenovirus.

In some embodiments, a composition of the presently claimed subjectmatter comprises a Bmx antagonist and a carrier to effect sustainedbioavailability of the Bmx antagonist following administration to asubject. Representative compositions for sustained bioavailability of aBmx antagonist can include but are not limited to polymer matrices,including swelling and biodegradable polymer matrices, (U.S. Pat. Nos.6,335,035; 6,312,713; 6,296,842; 6,287,587; 6,267,981; 6,262,127; and6,221,958), polymer-coated microparticles (U.S. Pat. Nos. 6,120,787 and6,090,925) a polyol:oil suspension (U.S. Pat. No. 6,245,740), porousparticles (U.S. Pat. No. 6,238,705), latex/wax coated granules (U.S.Pat. No. 6,238,704), chitosan microcapsules, and microsphere emulsions(U.S. Pat. No. 6,190,700).

An exemplary embodiment for sustained bioavailability of a Bmxantagonist comprises a gene therapy construct comprising a gene therapyvector, for example a gene therapy vector described herein below.

Viral Gene Therapy Vectors. In some embodiments, viral vectors of thepresently claimed subject matter are disabled; e.g.,replication-deficient. That is, they lack one or more functional genesrequired for their replication, which prevents their uncontrolledreplication in vivo and avoids undesirable side effects of viralinfection. In some embodiments, all of the viral genome is removedexcept for the minimum genomic elements required to package the viralgenome incorporating the therapeutic gene into the viral coat or capsid.For example, it is desirable to delete all the viral genome except: (a)the Long Terminal Repeats (LTRs) or Inverted Terminal Repeats (ITRs);and (b) a packaging signal. In the case of adenoviruses, deletions aretypically made in the E1 region and optionally in one or more of the E2,E3, and/or E4 regions. Other viral vectors can be similarly deleted ofgenes required for replication. Deletion of sequences can be achieved byrecombinant approaches, for example, involving digestion withappropriate restriction enzymes, followed by religation.Replication-competent self-limiting or self-destructing viral vectorscan also be used.

Nucleic acid constructs of the presently claimed subject matter can beincorporated into viral genomes by any suitable approach known in theart. Typically, such incorporation is performed by ligating theconstruct into an appropriate restriction site in the genome of thevirus. Viral genomes can then be packaged into viral coats or capsidsusing any suitable procedure. In particular, any suitable packaging cellline can be used to generate viral vectors of the presently claimedsubject matter. These packaging lines complement thereplication-deficient viral genomes of the presently claimed subjectmatter, as they include, for example by incorporation into theirgenomes, the genes that have been deleted from the replication-deficientgenome. Thus, the use of packaging lines allows viral vectors of thepresently claimed subject matter to be generated in culture.

Suitable packaging lines for retroviruses include derivatives of PA317cells, ψ-2 cells, CRE cells, CRIP cells, E-86-GP cells, and 293GP cells.Line 293 cells can be used with adenoviruses and adeno-associatedviruses.

Plasmid Gene Therapy Vectors. Certain of the Bmx antagonists of thepresently disclosed subject matter can also be encoded by a plasmid.Advantages of a plasmid carrier include low toxicity and easylarge-scale production. A polymer-coated plasmid can be delivered usingelectroporation as described by Fewell et al., 2001. Alternatively, aplasmid can be combined with an additional carrier, for example acationic polyamine, a dendrimer, or a lipid, that facilitates delivery(Baher et al., 1999; Maruyama-Tabata et al., 2000; Tam et al., 2000).

Liposomes. A Bmx antagonist of the presently claimed subject matter canalso be delivered using a liposome. For example, a nucleic acid moleculeencoding a Bmx antagonist can be encapsulated in a liposome. Liposomescan be prepared by any of a variety of techniques that are known in theart. See e.g., Dracopoli et al., 1997; Lasic & Martin, 1995; Janoff,1999; Gregoriadis, 1993; Betageri et al., 1993.; and U.S. Pat. Nos.4,235,871; 4,551,482; 6,197,333; and 6,132,766. Temperature-sensitiveliposomes can also be used, for example THERMOSOMES™, as disclosed inU.S. Pat. No. 6,200,598. Entrapment of a Bmx antagonist within liposomesof the presently claimed subject matter can be carried out using anyconventional method in the art. In preparing liposome compositions,stabilizers such as antioxidants and other additives can be used.

Other lipid carriers can also be used in accordance with the claimedpresently claimed subject matter, such as lipid microparticles,micelles, lipid suspensions, and lipid emulsions. See e.g., Labat-Moleuret al., 1996; and U.S. Pat. Nos. 5,011,634; 6,056,938; 6,217,886;5,948,767; and 6,210,707.

V.B. Targeting Ligands

As desired, a composition of the presently claimed subject matter caninclude one or more ligands having affinity for a specific cellularmarker to thereby enhance delivery of a Bmx antagonist to a targettissue, such as a tumor, in vivo. Ligands include antibodies, cellsurface markers, peptides, and the like, which act to home the Bmxantagonist to a tumor, including the tumor vasculature.

The terms “targeting” and “homing”, as used herein to describe the invivo activity of a ligand following administration to a subject, eachrefer to the preferential movement and/or accumulation of a ligand in atarget tissue (e.g., a tumor) as compared with a control tissue.

The term “control tissue” as used herein refers to a site suspected tosubstantially lack binding and/or accumulation of an administeredligand. For example, in some embodiments, a non-cancerous tissue can bea control tissue.

The terms “selective targeting” of “selective homing” as used hereineach refer to a preferential localization of a ligand that results insome embodiments in an amount of ligand in a target tissue that is about2-fold greater than an amount of ligand in a control tissue, in anotherembodiment in an amount that is about 5-fold or greater, and in stillanother embodiment in an amount that is about 10-fold or greater. Theterms “selective targeting” and “selective homing” also refer to bindingor accumulation of a ligand in a target tissue concomitant with anabsence of targeting to a control tissue, or the absence of targeting toall control tissues.

The terms “targeting ligand” and “targeting molecule” as used hereineach refer to a ligand that displays targeting activity. In someembodiments, a targeting ligand displays selective targeting.Representative targeting ligands include peptides and antibodies.

The term “peptide” encompasses any of a variety of forms of peptidederivatives that include amides, conjugates with proteins, cyclizedpeptides, polymerized peptides, conservatively substituted variants,analogs, fragments, peptoids, chemically modified peptides, and peptidemimetics. Representative peptide ligands that show tumor-bindingactivity include, for example, those described in U.S. Pat. Nos.6,180,084 and 6,296,832.

The term “antibody” indicates an immunoglobulin protein, or functionalportion thereof, including a polyclonal antibody, a monoclonal antibody,a chimeric antibody, a hybrid antibody, a single chain antibody (e.g., asingle chain antibody represented in a phage library), a mutagenizedantibody, a humanized antibody, and antibody fragments that comprise anantigen binding site (e.g., Fab and Fv antibody fragments).Representative antibody ligands that can be used in accordance with themethods of the presently claimed subject matter include antibodies thatbind the tumor-specific antigens Her2/neu (v-erb-b2 avian erythroblasticleukemia viral oncogene homologue-2; Kirpotin et al., 1997; Becerril etal., 1999) and antibodies that bind to CEA (carcinoembryonic antigen;Ito et al., 1991). See also U.S. Pat. Nos. 5,111,867; 5,632,991;5,849,877; 5,948,647; 6,054,561 and PCT International Publication No. WO98/10795.

In an effort to identify ligands that are capable of targeting tomultiple tumor types, targeting ligands have been developed that bind totarget molecules present on tumor vasculature (Baillie et al., 1995;Pasqualini & Ruoslahti, 1996; Arap et al., 1998; Burg et al., 1999;Ellerby et al., 1999).

A targeting ligand can also comprise a ligand that specifically binds toa radiation induced target molecule. Ionizing radiation induces proteinsin tumor vascular endothelium through transcriptional induction and/orposttranslational modification of cell adhesion molecules such asintegrins (Hallahan et al., 1995; Hallahan et al., 1996; Hallahan etal., 1998; Hallahan & Virudachalam, 1999). For example, radiationinduces activation of the integrin α_(2b)β₃, also called the fibrinogenreceptor, on platelets. The induced molecules can serve as binding sitesfor targeting ligands. A representative peptide ligand that binds toirradiated tumors comprises Bibapcitide (ACUTECT® available fromDiatide, Inc. of Londonberry, N.H., United States of America), whichspecifically binds to glycoprotein (GP) IIb/IIIa receptors on activatedplatelets (Hawiger et al., 1989; Hawiger & Timmons, 1992; Hallahan etal., 2001).

Antibodies, peptides, or other ligands can be coupled to drugs (e.g., aBmx antagonist) or drug carriers using methods known in the art,including but not limited to carbodiimide conjugation, esterification,sodium periodate oxidation followed by reductive alkylation, andglutaraldehyde crosslinking. See e.g., Bauminger & Wilchek, 1980;Dracopoli et al., 1997; Goldman et al., 1997; Kirpotin et al., 1997;Neri et al., 1997; Park et al., 1997; Pasqualini et al., 1997; U.S. Pat.No. 6,071,890; and European Patent No. 0 439 095. Alternatively,pseudotyping of a retrovirus can be used to target a virus towards aparticular cell (Marin et al., 1997).

A composition of the presently claimed subject matter comprises in someembodiments a Bmx antagonist and a pharmaceutically acceptable carrier.Suitable formulations include aqueous and non-aqueous sterile injectionsolutions, which can contain anti-oxidants, buffers, bacteriostats,bactericidal antibiotics, and solutes which render the formulationisotonic with the bodily fluids of the intended recipient; and aqueousand non-aqueous sterile suspensions which can include suspending agentsand thickening agents. The formulations can be presented in unit-dose ormulti-dose containers, for example sealed ampoules and vials, and can bestored in a frozen or freeze-dried (lyophilized) condition requiringonly the addition of sterile liquid carrier, for example water forinjections, immediately prior to use. Examples of useful ingredients aresodium dodecyl sulfate (SDS), for example in the range of 0.1 to 10mg/ml, in another example about 2.0 mg/ml; and/or mannitol or anothersugar, for example in the range of 10 to 100 mg/ml, in another exampleabout 30 mg/ml; phosphate buffered saline (PBS), and any otherformulation agents conventional in the art.

The therapeutic regimens and pharmaceutical compositions of thepresently claimed subject matter can be used with additional adjuvantsor biological response modifiers including, but not limited to, thecytokines interferon alpha (IFN-α), interferon gamma (IFN-γ),interleukin 2 (IL2), interleukin 4 (IL4), interleukin 6 (IL6), tumornecrosis factor (TNF), or other cytokine affecting immune cells.

VI. Therapeutic Methods

The presently disclosed subject matter also provides therapeutic methodsthat can be employed for treating and/or otherwise ameliorating one ormore symptoms and/or consequences of undesirable underexpression orundesirable overexpression of a Bmx gene product.

In some embodiments, the presently disclosed subject matter providesmethods for modulating proliferation of a cell or of a tissue (e.g., atumor) in a subject, the methods comprising administering to the subjectan effective amount of a modulator of a biological activity of a bonemarrow X kinase (Bmx) gene product.

In some embodiments, the presently claimed subject matter providesmethods for suppressing tumor growth comprising (a) administering a Bmxantagonist to a subject bearing a tumor to increase the radiosensitivityof the tumor; and (b) treating the tumor with ionizing radiation,whereby tumor growth is delayed. Also provided is a method forinhibiting tumor blood vessel growth via administration of a Bmxantagonist.

In some embodiments, the presently disclosed subject matter providesmethods for inhibiting tumor blood vessel growth comprising (a)administering a bone marrow X kinase (Bmx) antagonist, a vector encodinga bone marrow X kinase (Bmx) antagonist, or a combination thereof to asubject bearing a tumor to increase the radiosensitivity of tumor bloodvessels; and (b) treating the tumor with ionizing radiation, wherebytumor blood vessel growth is inhibited.

While applicants do not intend to be bound by any particular theory ofoperation, it is believed that the Bmx antagonists disclosed hereineffectively suppress proliferation of a cell or of a tissue (e.g., atumor growth) by blocking perfusion of the tissue (e.g., reperfusion ofa tumor subsequent to and/or concurrent with radiation). Specifically, aBmx antagonist can block processes that require Bmx, including themediation of growth factor signals that result in endothelial cellinfiltration and budding of tumor blood vessels. Similarly, a Bmxantagonist is believed to effectively inhibit the growth of tumor bloodvessels by blocking the ability of growth factors to mediate bloodvessel growth.

In some embodiments, the presently disclosed subject matter providesmethods for inhibiting a condition associated with undesirableangiogenesis in a subject, the method comprising administering to thesubject an effective amount of a bone marrow X kinase (Bmx) antagonist.As used herein, the phrase “condition associated with undesirableangiogenesis” refers to any condition at least one symptom of which iscaused by the presence in a subject of a vascular network that isabnormal in some clinically relevant manner including, but not limitedto the extent to which the vascular network develops and/or the timeand/or place at which it develops. In some embodiments, a conditionassociated with undesirable angiogenesis is macular degeneration orendometriosis.

As would be understood by one of ordinary skill in the art, angiogenesisis the process whereby new blood vessels are formed. Angiogenesis, alsocalled neovascularization, occurs normally during embryogenesis anddevelopment, and occurs in fully developed organisms during woundhealing and placental development. In addition, angiogenesis occurs invarious pathological conditions, including in ocular diseases such asdiabetic retinopathy and macular degeneration due to neovascularization,in conditions associated with tissue inflammation such as rheumatoidarthritis and inflammatory bowel disease, in cancer, where blood vesselformation in the growing tumor provides oxygen and nutrients to thetumor cells, as well as providing a route via which tumor cellsmetastasize throughout the body, and in endometriosis.

Angiogenesis occurs in response to stimulation by one or more knowngrowth factors, and can also involve other as yet unidentified factors.Endothelial cells, which are the cells that line mature blood vessels,normally do not proliferate. However, in response to an appropriatestimulus, the endothelial cells become activated and begin toproliferate and migrate into unvascularized tissue to form new bloodvessels. In some cases, precursor cells can be activated todifferentiate into endothelial cells, which form new blood vessels.

In pathological conditions such as age-related macular degeneration anddiabetic retinopathy, decreasing availability of oxygen to the retinaresults in a hypoxic condition that stimulates the secretion ofangiogenic growth factors such as vascular endothelial growth factors(VEGF), which induce abnormal migration and proliferation of endothelialcells into tissues of the eye. Such vascularization in ocular tissuescan induce corneal scarring, retinal detachment, and fluid accumulationin the choroid, each of which can adversely affect vision and lead toblindness.

Angiogenesis also is associated with the progression and exacerbation ofinflammatory diseases, including psoriasis, rheumatoid arthritis,osteoarthritis, and inflammatory bowel diseases such as ulcerativecolitis and Crohn's disease. In inflammatory arthritic disease, forexample, influx of lymphocytes into the region surrounding the jointsstimulates angiogenesis in the synovial lining. The increasedvasculature provides a means for greater influx of leukocytes, whichfacilitate the destruction of cartilage and bone in the joint.Angiogenic vascularization that occurs in inflammatory bowel diseaseresults in similar effects in the bowel.

The growth of capillaries into atherosclerotic plaques in the coronaryarteries represents another pathological condition associated withgrowth factor induced angiogenesis. Excessive blood flow intoneovascularized plaques can result in rupture and hemorrhage of theblood-filled plaques, releasing blood clots that can result in coronarythrombosis.

The involvement of angiogenesis in such diverse diseases as cancer,ocular disease, and inflammatory diseases has led to an effort toidentify methods for specifically inhibiting angiogenesis as a means totreat these diseases. For cancer patients, such methods of treatment canprovide a substantial advantage over currently used methods such aschemotherapy, which kill or impair not only the target tumor cells, butalso normal cells in the patient, particularly proliferating normalcells such as blood cells, epithelial cells, and cells lining theintestinal lumen. Such non-specific killing by chemotherapeutic agentsresults in side effects that are, at best, unpleasant, and can oftenresult in unacceptable patient morbidity, or mortality. In fact, theundesirable side effects associated with cancer therapies often limitthe treatment a patient can receive.

VI.A. Administration of a Bmx Antagonist

Suitable methods for administration of a composition of the presentlyclaimed subject matter include but are not limited to intravascular,subcutaneous, intramuscular, intraperitoneal, or intratumoraladministration. For delivery of compositions to pulmonary pathways,compositions can be administered as an aerosol or coarse spray. Adelivery method is selected based on considerations such as the type ofBmx antagonist, the type of carrier or vector, toxicity of the Bmxantagonist, therapeutic efficacy of the Bmx antagonist, and thecondition of the tumor to be treated. In some embodiments of thepresently claimed subject matter, intravascular administration isemployed.

In some embodiments, an effective amount of a composition of thepresently claimed subject matter is administered to a subject. An“effective amount” is an amount of a composition comprising a Bmxantagonist sufficient to produce a measurable response, such as but notlimited to an anti-tumor response (e.g., increase of radiationsensitivity, an anti-angiogenic response, a cytotoxic response, and/ortumor regression). Actual dosage levels of active ingredients in atherapeutic composition of the presently claimed subject matter can bevaried so as to administer an amount of the active compound(s) that iseffective to achieve the desired therapeutic response for a particularsubject. The selected dosage level will depend upon a variety of factorsincluding the activity of the therapeutic composition, formulation, theroute of administration, combination with other drugs or treatments,tumor size and longevity, and the physical condition and prior medicalhistory of the subject being treated. Determination and adjustment of atherapeutically effective dose, as well as evaluation of when and how tomake such adjustments, are known to those of ordinary skill in the artof medicine.

In some embodiments of the presently claimed subject matter, a minimallytherapeutic dose of a Bmx antagonist is administered. The term“minimally therapeutic dose” refers to the smallest dose, or smallestrange of doses, determined to be a therapeutically effective amount asdefined herein above.

VI.B. Radiation Treatment

For treatment of a radiosensitized target tissue, the target tissue isirradiated concurrent with, or subsequent to, administration of acomposition comprising a Bmx antagonist. In accordance with the methodsof the presently claimed subject matter, the target tissue can beirradiated daily for 2 weeks to 7 weeks (for a total of 10 treatments to35 treatments). Alternatively, target tissues can be irradiated withbrachytherapy utilizing high dose rate or low dose rate brachytherapyinternal emitters.

Subtherapeutic or therapeutic doses of radiation can be used fortreatment of a radiosensitized target tissue as disclosed herein. Insome embodiments, a subtherapeutic or minimally therapeutic dose (whenadministered alone) of ionizing radiation is used. For example, the doseof radiation can comprise at least about 2 Gy ionizing radiation, inanother example about 2 Gy to about 6 Gy ionizing radiation, and in yetanother example about 2 Gy to about 3 Gy ionizing radiation. Whenradiosurgery is used, representative doses of radiation include about 10Gy to about 20 Gy administered as a single dose during radiosurgery orabout 7 Gy administered daily for 3 days (about 21 Gy total). When highdose rate brachytherapy is used, a representative radiation dosecomprises about 7 Gy daily for 3 days (about 21 Gy total). For low doserate brachytherapy, radiation doses typically comprise about 12 Gyadministered twice over the course of 1 month. ¹²⁵I seeds can beimplanted into a target tissue and can be used to deliver very highdoses of about 110 Gy to about 140 Gy in a single administration.

Radiation can be localized to a target tissue using conformalirradiation, brachytherapy, stereotactic irradiation, or intensitymodulated radiation therapy (IMRT). The threshold dose for treatment canthereby be exceeded in the target tissue but avoided in surroundingnormal tissues. For treatment of a subject having two or more targettissues, local irradiation enables differential drug administrationand/or radiotherapy at each of the two or more target tissues.Alternatively, whole body irradiation can be used, as permitted by thelow doses of radiation required following radiosensitization of thetarget tissue.

Radiation can also comprise administration of internal emitters, forexample ¹³¹I for treatment of thyroid cancer, NETASTRON™ and QUADRAGEN®pharmaceutical compositions (Cytogen Corp. of Princeton, N.J., UnitedStates of America) for treatment of bone metastases, and ³²P fortreatment of ovarian cancer. Other internal emitters include ¹²⁵I,iridium, and cesium. Internal emitters can be encapsulated foradministration or can be loaded into a brachytherapy device.Radiotherapy methods suitable for use in the practice of the presentlydisclosed subject matter can be found in Leibel & Phillips, 2004, amongother sources.

EXAMPLES

The following EXAMPLES provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following EXAMPLES are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlydisclosed subject matter.

Materials and Methods for the Examples

Cell culture. HUVECs were purchased from Clonetics Corp. (San Diego,Calif., United States of America) and maintained in EBM-2 mediumsupplemented with EBM-2 singlequots (Cambrex Corp., East Rutherford,N.J., United States of America). HUVECs were limited to passages 3-6.Lewis Lung carcinoma (LLC) cells were purchased from American TypeCulture Collection (Manassas, Va., United States of America) andmaintained in DMEM supplemented with 10% FBS and 1%penicillin-streptomycin. Cell lines were incubated at 37° C. in a 5% CO₂incubator.

LFM-A13 (30 μM in vitro or 50 mg/kg, intraperitoneal (i.p.) in vivo) andDMSO was obtained from Sigma-Aldrich Co. (St. Louis, Mo., United Statesof America). Drug was administered to cells 30-60 min prior irradiation.A Mark-1 Irradiator ¹³⁷Cs (JL Shepard and Assoc., San Fernando, Calif.,United States of America) was used to irradiate HUVEC cultures at a doserate of 1.897 Gy/min.

Retrovirus production and HUVEC infection. Negative control and BmxshRNA retroviral constructs were purchased from OriGene Inc (Rockville,Md., United States of America). A total of 5 different constructs(labeled Bmx A-E) were tested for Bmx knockdown in HUVEC. Theretroviruses were produced according to manufacturer's protocol withsome modifications. LiNX packaging cell line, purchased from OpenBiosystems Inc. (Huntsville, Ala., United States of America), was grownon 10 cm tissue culture plates to 30-40% confluency in media containingDMEM with 10% fetal bovine serum (referred to herein as Complete GrowthMedium (CGM)) with hygromycin, penicillin, and streptomycinsupplementation. These cells were then transfected with retroviralplasmid DNA by incubation with 5 ml transfection mix for 4-6 hours. Thetransfection mix contained 12 μg of shRNA retroviral vector DNA within600 μl serum-free DMEM without antibiotics, 240 μl of room temperatureArrest-In transfection reagent (Open Biosystems Inc.) within 4.4 mlserum-free DMEM without antibiotics which was prepared and kept at roomtemperature for 45 minutes prior to transfection to allow fortransfection complexes to be formed. Following initially 4-6 hourstransfection, 5 ml of CGM was added and incubated overnight. The mediawas changed and the cells were incubated for at least an additional 24hours. Supernatant was collected and filtered through a 45 μm filter toproduce the viral stock.

For HUVEC infection, HUVEC were grown on tissue culture plates to 50%confluency. On the day of infection, the HUVEC was incubated in mediumcontaining 5 μg/ml polybrene for 4 hours prior to infection. Medium wasremoved and 1.5 ml of virus supernatant supplemented with 5 μg/mlpolybrene was added directly to the cells and allowed to adsorb for40-60 minutes. 7 ml of HUVEC media containing 5 μg/ml of polybrene wasadded and the cells are incubated for 24 hours. The media was changed toregular HUVEC growth media and the cells were incubated for anadditional 2 days to allow for Bmx knockdown.

Cell lysis and immunoblot analysis. HUVECs of passage 3-6 were treatedwith or without Bmx inhibition (LFM-A13 for 60 minutes or shRNAretrovirus infection 48 hours prior) followed by irradiation and thenharvested at the indicated times. Cells were processed and immunoblottedas described in Cuneo et al., 2007. Antibodies were the following:PY20HRP (BD Biosciences, San Jose, Calif., United States of America),actin (Sigma-Aldrich Co.), Bmx and PY40 Bmx (Cell Signaling Technology,Danvers, Mass., United States of America), phospho-Akt (S473), Akt (CellSignaling Technology) as well as HRP labeled mouse anti-rabbit secondaryantibodies (Sigma-Aldrich) except for PY20HRP which was pre-labeled.Films were scanned into Adobe Photoshop with subsequent densitometryanalysis. Experiments were performed at least three times.

Immunoprecipitation and in vitro kinase assay. HUVECs of passage 3-6were grown to 70-80% confluency and then serum starved for 5 hours. Thecells were then treated with sham or 3 Gy irradiation. After treatment,the cells were incubated at 37° C. with 5% CO₂ for the indicated times.For inhibition studies, inhibitor was added at a 1:1000 dilution 60minutes prior to irradiation. Following incubation, the tissue culturedishes were placed on ice and washed twice with ice cold 1×Phosphate-buffered Saline (PBS) followed by lysis using 400 μM-PERcontaining protease and phosphatase inhibitors (Sigma-Aldrich Co.) for 5minutes. The cells were scraped and transferred to Eppendorf tubes,vortexed for 20 seconds, and incubated on ice for 30 minutes. Afterclearing the lysate by centrifugation at 15,000 g for 15 minutes at 4°C., the supernatant was quantified for protein concentration using thebicinchoninic acid (BCA) method prior to immunoprecipitation.Immunoprecipitation was performed using the CATCH AND RELEASE® v 2.0system (Upstate Group, LLC, Charlottesville, Va., United States ofAmerica) according to manufacturer's protocol with some modification.

Briefly, spin columns containing binding resin were prepared by washingtwice with 1× wash buffer (2000 g/30 seconds). After column preparation,500 μg of cell lysate was combined with 2 μg of anti-Bmx antibody (SantaCruz Biotechnology, Inc., Santa Cruz, Calif., United States of America),10 μl of affinity ligand, and enough 1× wash buffer to have a 500 μlfinal volume. This was added to the capped spin column which was rotatedend-over-end overnight at 4° C. The spin column was washed thrice with1× wash buffer followed by elution using 70 μl of 1× non-denaturingelution (native protein elution) buffer. For in vitro kinase assay, 35μl of eluate was then combined with 25 μl of kinase buffer containing 25mM Tris (pH 7.5), 5 mM β-Glycerophosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10mM MgCl₂ with 1 μl of 10 mM ATP and this was incubated for 20 minutes at37° C. The reaction was stopped by adding 20 μl of 4×SDS sample bufferand boiling for 5 minutes. Samples were then run on SDS-PAGE andanti-PY20HRP (BD Biosciences) Western blotting was performed to identifyphosphorylated protein bands at 75-80 kilodaltons (kDa). Blots were thenstripped and re-probed for total Bmx.

WST-1 assay. This assay is a modification of an MTT assay and wasperformed per the manufacturer's protocol (Calbiochem, San Diego,Calif., United States of America). Briefly, HUVEC were infected withshRNA retrovirus and after 48 hours incubation, cells were lifted bytrypsinization, counted, and plated at 10,000 cells/well of 96-wellmicrotiter plates in duplicate. These plates were then subjected to 0 or2 Gy and incubated for 24 hours at 37° C. with 5% CO₂. Followingincubation, 10 μl of WST-1 labeling mixture was added to each well andmixed gently prior to returning to the incubator for 2 hours. Opticaldensity at 450 nm (OD₄₅₀ nm) was measured on a microplate reader andresults were plotted using Microsoft Excel Software (Microsoft Corp.,Redmond, Wash., United States of America).

Clonogenic survival. LLC's or passage 3-6 HUVECs were grown to 70-80%confluency. Cells were washed with 1×PBS, trypsin suspended, and werecounted and adjusted to specific densities for each condition. The cellswere plated on tissue culture plates and allowed to attach for 4 hours.LFM-A13 or DMSO was added at a 1:1000 dilution followed 60 min later by0, 2, 4 or 6 Gy. Media was changed after irradiation. 10-14 days afterirradiation, the plates were fixed with 70% ethanol and stained with 1%methylene blue. Colonies were then counted using a dissection microscopywith positive colonies containing at least 50 cells. Surviving fraction(SF) was calculated by the equation (number of colonies formed/number ofcells plated)/(number of colonies for sham irradiated group/number ofcells plated). Dose enhancing ratios were calculated by dividing thedose (Gy) for radiation alone by the dose for radiation plus treatment(normalized for plating efficiency of treatment) for which a SF of 0.2is achieved. These results were then plotted in a semilogarithmic formatusing Microsoft Excel software (Microsoft Corp.).

Endothelial cell tubule formation assay. HUVEC of passage 3-6 were grownto 70-80% confluency. Cells were then washed with 1×PBS and suspended bytrypsinization. Cells were counted and adjusted to 2.5-5×10⁴ cells/ml inmedia. 75 μl MATRIGEL™ (BD Biosciences) were plated into each well of a96 well plate and allowed to polymerize at 37° C. Cell suspensions (200μl; 8-12×10³ cells) were added to each well. After thirty minutes, DMSOcontrol or LFM-A13 was added. Thirty minutes later, dishes were treatedwith sham or 3 Gy and were then incubated until tubules had formed incontrol plates (4-6 hours). Digital photographs were taken of individualwells and tubules were counted by an observer blinded to the treatmentconditions. The mean and standard error were calculated (n=3).

Endothelial closure assay. HUVEC of passage 3-6 were grown on glassslides and were subjected to gap formation as described previously(Cuneo et al., 2007). Cells were treated with LFM-A13 or DMSO controlprior to 0 or 3 Gy and incubated for the indicated times. Photographs ofcell defect and surrounding cells were taken and relative cell densitywithin the defect was calculated as follows: (number of cells/originalcell defect area)/(number of cells/surrounding area).

Apoptosis assays. HUVEC were grown to 70-80% confluency prior totreatment with LFM-A13 or DMSO control. Cells were then irradiated with0 or 3 Gy and harvested 24 hours after irradiation. Annexin V-FITCApoptosis Detection kit (BD PharMingen, San Diego, Calif., United Statesof America) was used to stain cells (propidium iodide (50 ng) andannexin V-FITC (5 ng) were added to 10⁵ cells) for flow cytometryaccording to manufacturer's protocol. For each treatment, the percent ofcells undergoing apoptosis (±SE) was calculated. Camptothecin (5 μmol/L)treatment for 6 hours served as positive control

HUVEC were also assayed for apoptosis using a4′,6-Diamidino-2-phenylindole (DAPI) staining of the nuclei to identifycells undergoing morphological changes. 70%-80% confluent HUVECs weretreated with or without LFM-A13, incubated for 1 hours, and thenirradiated at 3 Gy. Cells were returned to the incubator for anadditional 24 hr prior to DAPI staining. Multiple high powered fields(at least 7) were examined by an observer who was blinded to theexperimental conditions for each of the cultures. The percentage ofcells demonstrating apoptotic nuclei were quantified. The mean andstandard error were calculated for each treatment group.

Tumor vascular window model. The tumor vascular window model techniqueis described in Edwards et al., 2002. Briefly, three mice for each grouphad tumors grown within a vascular window such that tumor vasculaturecould be visualized within window frames containing a coordinate systemfor serial photography. Animals were treated with LFM-A13 by i.p.injection 60 minutes prior to 2 Gy irradiation using an 80 kVpsuperficial X-ray machine (Pantak X-ray Generator; East Haven, Conn.,United States of America). Serial color photographs were taken todocument blood vessel appearance on days 0-7. Photographs were scannedand processed using ADOBE® PHOTOSHOP® software (Adobe Systems Inc., SanJose, Calif., United States of America) to mark the center of vessels,verified by an observer blinded to treatment groups. Vascular lengthdensity (VLD) was quantified for each microscopic field using IMAGE-PRO®Plus v. 5.1 software (Media Cybernetics, Inc. Silver Spring, Md., UnitedStates of America). The mean and standard error of VLD in each treatmentgroup were calculated and plotted. All animals used were cared foraccording to Vanderbilt University's Institutional Animal Care and UseCommittees guidelines.

Tumor immunohistochemistry. LLC's were subcutaneously injected into thehind limb of C57BL/6 mice to form xenografts. When tumors grew to 200mm³, (approximately 7 days) the mice were treated with five consecutivedaily treatments of i.p. LFM-A13 followed 45 minutes later by 3 Gyirradiation using an 80 kVp superficial x-ray generator. Twelve hoursfollowing the last radiation treatment, mice were sacrificed and tumorswere harvested, fixed in paraffin, and sectioned by the VanderbiltUniversity Immunohistochemistry Core Facility as described previously(see Cuneo et al., 2007). Immunostaining was with goat anti-CD34 (SantaCruz Biotechnology) and microvascular photos were analyzed usingIMAGE-PRO® software with pixel number quantified.

Tumor growth delay. LLC's were subcutaneously injected into the hindlimb of C57/BL6 mice to form xenografts. When tumors grew to 200 mm³,(approximately 7 days) the mice were treated with five consecutive dailytreatments of i.p. LFM-A13 followed 45 minutes later by 3 Gy irradiationusing an 80 kVp superficial x-ray generator. Serial measurements oftumor dimensions were taken by caliper and tumor volume was calculatedusing the modified ellipsoid formula (length×width×depth)/2. The meanand standard error were plotted using Microsoft Excel software.

Statistical analysis. The mean and standard errors for all assays werecalculated using Microsoft Excel software. Student's t test wasperformed to determine P values between treatment groups. P values lessthan or equal to 0.05 were considered statistically significant.

Example 1 Bmx is Activated in Endothelium Upon Irradiation

Primary culture vascular endothelial cells (HUVEC) were examined todetermine whether Bmx was activated by IR because of its similarities instructure and signaling to the serine/threonine protein kinase Akt/PKB,and also whether it contributed to radiation resistance. FIG. 1A depictsanalysis of a time course of Bmx activation upon irradiation with aclinically relevant dose of 2 Gy. Tyrosine 40, present in the PH domainof Bmx, becomes phosphorylated during its activation (Chen et al.,2001). As shown in FIG. 1A, Bmx was phosphorylated at 60 minutesfollowing 2 Gy of irradiation.

To confirm this result, an in vitro kinase (IVK) assay was employed inwhich Bmx was immunoprecipitated from irradiated or sham irradiatedendothelial cells and then incubated with ATP in a kinase reaction.These samples were run on SDS-PAGE and probed with ananti-phosphotyrosine antibody to analyze autophosphorylation of Bmx. Asshown in FIG. 1B, Bmx was activated after irradiation. Interestingly,Bmx showed significant kinase activity immediately following irradiationand then has a second peak of activity at 1 hour.

Example 2 Bmx Knockdown Enhanced Radiation Efficacy in Endothelium

Because a clear activation of Bmx following a clinically relevant doseof IR was observed, whether Bmx activation protected the endothelialcells from cytoxic damage was examined. Since primary cultureendothelial cells, such as HUVEC, have low transfection yields, aretroviral shRNA system was employed to knockdown Bmx levels prior toirradiation. FIG. 2A shows five different retroviral constructs (Athrough E) for Bmx as well as a negative control construct (Neg) thatwere used to infect HUVEC. After 48 hours, infected cells were harvestedand lysates were prepared for total Bmx Western blotting. As can beseen, construct A (shBmxA) provided about a 90% knockdown of Bmx proteinlevels compared to the negative control shRNA vector. Bmx knockdownexperiments were performed with or without irradiation.

FIG. 2B shows the results of MTT-based (WST-1) survival assays. HUVECwere infected with either shBmxA or negative control vectors. After 48hours, cells were counted and plated at 10,000 cells/well in duplicatewithin 96-well dishes. The cells were treated with either sham (0 Gy) or2 Gy irradiation and incubated for 24 hours. Following this incubation,WST-1 labeling mixture was added to each well and analyzed at todetermine mitochondrial viability by optical density at 45 nm (OD₄₅₀).Normalized values for OD₄₅₀ are shown as mean and standard error.Combined Bmx knockdown with irradiation decreased HUVEC survival.

Example 3 Pharmacological Inhibition of Bmx

Having established that Bmx knockdown enhanced radiation efficacy inendothelial cells, whether or not pharmacological inhibition of Bmxwould show the same effect was examined. Bmx specific inhibitors havebeen described (see e.g., Kawakami et al., 1999; Mahajan et al., 1999;Uckun et al., 2002; He et al., 2004), particularly LFM-A13, whichtargets the Tec family. Since Bmx is the only Tec family memberexpressed in endothelium, this drug was studied in HUVEC. LFM-A13 hasbeen shown to block VEGF induced signaling through Bmx inhibition inHUVEC at a dose of 25 μM. Therefore, 30 μM LFM-A13 was employed for invitro studies. FIG. 3A shows the use of vehicle control (DMSO) or 30 μMLFM-A13 pre-incubation on radiation-induced Bmx activation in the invitro kinase assay. As can be seen, LFM-A13 attenuated the activation ofBmx in response to 3 Gy.

Example 4 Bmx Inhibition Attenuated Endothelial Cell Viability

To determine whether LFM-A13 produces a radiosensitization effect inHUVEC, clonogenic survival assays in HUVEC with LFM-A13 pre-incubationwas studied (see FIG. 3B). HUVEC were pre-treated with DMSO vehiclecontrol or 30 μM LFM-A13 for 45 minutes prior to irradiation with 0, 2,4, or 6 Gy. Colonies were allowed to form over 10 days, which were thencounted and the surviving fraction was calculated for each radiationdose. These studies indicated that 30 μM LFM-A13 radiosensitized HUVECcompared to the control as evidenced by the downward survival curveshift. The dose enhancing ratio (DER) was 1.47.

Apoptosis was studied to determine whether this was a mechanism ofenhanced cytotoxicity. FIG. 3C illustrates the effect of LFM-A13 onapoptosis within these cells. HUVEC treated with 30 μM LFM-A13 or DMSOcontrol were subjected to sham or 3 Gy irradiation and then incubatedfor 24 hours prior to trypsinization and flow cytometric analysis.Annexin V-propidium iodide staining revealed that drug or 3 Gy alone wasnot capable of shifting cells into either early (Q4-1) or late (Q2-1)apoptosis, but that the combination of LFM-A13 and 3 Gy caused astatistically significant (p<0.001 vs. LFM-A13 or 3 Gy alone) increasein apoptosis. To confirm these findings, HUVEC were treated with either30 μM LFM-A13 or DMSO control with or without 3 or 6 Gy irradiation andincubated for 24 hours. The cells were fixed and stained with DAPI andthe percent of apoptotic cells was quantified. As shown in FIG. 3D, thecombination of LFM-A13 and irradiation resulted in enhancement ofapoptosis. In FIG. 3D, * indicates p<0.05 vs. DMSO control and **indicates p<0.001 vs. LFM alone.

Example 5 Bmx Inhibition Attenuated Endothelial Cell Function

Functional assays of endothelial cells include cell migration andcapillary-like tubule formation. FIG. 4A illustrates the effect ofLFM-A13 and irradiation on endothelial migration across a gap(endothelial cell closure assay) at 12 and 24 hours. HUVEC were platedon glass slides and grown to 80% confluency. A gap region (i.e., aregion on the glass slide that was made free of cells) was then createdusing a 200 μl pipette tip. The slides were then treated with 30 μMLFM-A13 or DMSO control for 45 minutes prior to either 0 or 3 Gy. Cellswere fixed and stained at 12 or 24 hours and photographs were taken ofthe gap region and the surrounding cells to determine the ability of theHUVEC to migrate across and fill the gap. Relative cell density wascalculated for each condition to control for the cytotoxic effects oftreatment as shown in FIG. 4B. By 24 hours, control cells effectivelymigrated across the gap. 30 μM LFM-A13 or 3 Gy alone had minimal effecton attenuating endothelial cell closure at both 12 hours and 24 hourscompared to vehicle treated control. However, the combination induced agreater than additive effect which was statistically significant (*indicates p<0.05 vs. control, and ** is p<0.01 vs. LFM-A13).

FIGS. 4C and 4D show the results of capillary tubule formation assays.HUVEC plated onto a MATRIGEL™ matrix were treated with 30 μM LFM-A13 orDMSO with or without 3 Gy irradiation and allowed to form tubules. Thecells were then fixed and stained. The number of tubules was quantifiedand plotted. Representative photographs are shown in FIG. 4C andquantified in FIG. 4D. Cells that were treated with both LFM-A13 and 3Gy showed a significant reduction (p<0.005) in tubules formed comparedto cells treated with either treatment alone.

Example 6 Bmx Inhibition Attenuated Tumor Vasculature

To determine whether Bmx inhibition enhanced radiation-induceddestruction of tumor vasculature in vivo, intraperitoneal (i.p.)injection of LFM-A13 was utilized prior to irradiation. A tumor vascularwindow chamber was placed on the dorsum of C57BL/6 mice and Lewis LungCarcinoma (LLC) cells were implanted within the dorsal skin fold toallow for visualization of intravital tumor vasculature. Serialphotographs were taken of the same region of the tumor allowing forassessment of blood vessel formation and destruction. FIG. 5A shows theeffect of a single 50 mg/kg i.p. administration of LFM-A13 prior to 2 Gyirradiation. Representative photographs show that combination treatmentresulted in a dramatic reduction in tumor blood vessels. These resultswere quantified for each treatment condition as mean vascular lengthdensity with standard error (see FIG. 5B; p<0.0014 vs. LFM-A13 or 2 Gyalone).

To confirm these results, a hind limb xenograft model was employed fordetermining vascular density within tumor sections. LLCs were implantedinto the hind limbs of C57BL/6 mice. After tumors were formed, they weresubjected to either daily LFM-A13 (50 mg/kg i.p. injection) or DMSOfollowed 45 minutes later by 3 Gy or sham irradiation for a total offive treatments. The tumors were then harvested and prepared forimmunohistochemistry analysis. Vessels were stained by anti-CD34 asshown in FIG. 5C and these were quantified as shown in FIG. 5D. As canbe seen, combination treatment was most effective at attenuating bloodvessel formation (p=0.043 vs. IR; p=0.0001 vs. LFM-A13 or vehiclecontrol).

Example 7 Bmx Inhibition did not Affect Radiation Sensitivity of LLC

Whether Bmx inhibition could affect radiation sensitization in the LewisLung Carcinoma (LLC) cell line was also tested. As shown in FIG. 6A,LFM-A13 showed no difference in clonogenic survival compared to DMSOcontrol in LLCs, suggesting that LFM-A13 enhancement of radiationpertained to its anti-vascular effect in this tumor model, although itis not desired to be bound by any particular theory of operation.

Example 8 Bmx Inhibition Enhanced Radiation Efficacy in Tumor GrowthDelay

Although LFM-A13 treatment did not affect the radiosensitivity of theLLCs, LFM-A13 could still enhance radiation effects in vivo as ananti-vascular treatment. To determine whether treatment with LFM-A13could enhance tumor growth delay in irradiated tumors, mice bearing LLChind limb tumors were treated as in FIG. 5C, with i.p. injection of 50mg/kg LFM-A13 or DMSO 45 minutes prior to 3 Gy or sham irradiation for 5consecutive days. The mean tumor volume and standard error are plottedfor each treatment group in FIG. 6B. Whereas LFM-A13 or radiationtreatment alone resulted in a small growth delay, combination treatmentdemonstrated a statistically significant enhancement of growth delay(p=0.027), suggesting that LFM-A13 can enhance the efficacy oftherapeutic radiation.

Discussion of the Examples

Disclosed herein are investigations into the roles of Bmx in theradiation response of tumor vasculature. Activation of Bmx occurred atclinically relevant doses of radiation: 2-3 Gy. shRNA knockdown of Bmxresulted in radiation sensitization in HUVEC, suggesting that Bmxinhibition is a promising pharmacological target for radiationenhancement.

Although LMF-A13 is clinically used as a Btk inhibitor, many groups haveutilized LFM-A13 as a Bmx inhibitor due to the high homology between Bmxand Btk. Since Btk is only found in bone marrow derived cells, it wasbelieved that LFM-A13 could be used effectively for Bmx inhibition inthe model systems disclosed herein. As demonstrated in the in vitrokinase assays disclosed herein, LFM-A13 effectively attenuated Bmxactivation in response to radiation. This drug not only enhanced thecytotoxic effects of irradiation on HUVEC, but also inhibited thefunction of these cultured endothelial cells. Apoptosis and clonogenicstudies revealed that LFM-A13 was capable of inducing radiosensitizationin HUVEC. Moreover, LFM-A13 in combination with radiation resulted indramatic effects on endothelial cell migration as evidenced by theendothelial closure assay and tubule formation assay.

The vascular effects were more pronounced in the in vivo tumor models.Tumor vascular window blood vessels were minimally inhibited by 2 Gy orLFM-A13 alone. However, in combination, LFM-A13 and 2 Gy substantiallydisrupted tumor blood vessel formation. This anti-vascular effect wasconfirmed in hind limb tumor models, which showed that daily LFM-A13 and3 Gy significantly affected the tumor microvasculature. Tumor growthdelay was displayed in the combination arm which was more than additive.

Tec family kinase inhibition has garnered attention, although mainly inrelation to anti-inflammation. Interestingly, ImClone Systems Inc. hasdeveloped a Bmx single chain intrabody system which can partiallyattenuate Src transformation potential (Paz et al., 2005). Recently, Panet al. described a number of selective irreversible Btk inhibitors aimedat treating rheumatoid arthritis (Pan et al., 2007). Moreover, CGIPharmaceuticals, Inc. has been developing their own novel Btkinhibitors, cgi1316 and cgi1746, for use in inflammatory diseases.However, LFM-A13, a rationally designed inhibitor developed by theParker Hughes Institute, has been extensively tested in pre-clinicalmodels. The pharmacokinetics and toxicity data have been previouslypublished (Uckun et al., 2002) which provided the basis for the presentstudy. The drug appears to be well-tolerated based on these murinestudies. Another commercially available Tec family inhibitor, terreicacid (Kawakami et al., 1999), seemed to be at least as effective asLFM-A13 in the in vitro studies disclosed herein. However, since verylittle is known about in vivo toxicity and pharmacokinetics for terreicacid, the presently disclosed investigations focused on LFM-A13.

Bmx provides an alternative cell survival pathway to that of PI3K-Aktsignal transduction. It is possible that treatments that have targetedPI3K-Akt signaling might be deriving some of their efficacy fromconcomitant Bmx inhibition. Further studies can determine whether Bmxinhibition in combination with PI3K-Akt blockade provide additionalbenefit. Nevertheless, Bmx inhibition remains an attractive potentialtarget for radiation enhancement because Bmx activation occurs rapidlyand transiently following radiation such that prolonged Bmx inhibitionis probably not necessary for radiation sensitization to occur, which isconsistent with the cell culture assays disclosed herein in which thedrug was removed shortly after irradiation. Therefore, short acting drugformulations together with radiation are candidates for causing lesssystemic effects than typically occur with long-term administration.

In summary, Bmx is a new molecular target for radiation sensitizationbased on in vitro and in vivo experimentation in vascular endothelium.

REFERENCES

All references listed in the instant disclosure, including but notlimited to all patents, patent applications and publications thereof,scientific journal articles, and database entries (including but notlimited to GENBANK® database entries including all annotations availabletherein) are incorporated herein by reference in their entireties to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

-   Abassi et al. (2003) J Biol Chem 278:35636-35643.-   Adelman et al. (1983) DNA 2:183-193.-   Advani et al. (1998) Gene Ther 5:160-165.-   Allam et al. (1993) Intl J Radiat Oncol Biol Phys 27:303-308.-   Antonakopoulos et al. (1994) Histopathology 25:447-454.-   Arap et al. (1998) Science 279:377-380.-   Ausubel (ed.) (1995) Short Protocols in Molecular Biology, 3rd ed.    Wiley, New York, United States of America.-   Ausubel et al. (eds.) (1992) Current Protocols in Molecular Biology.    Wiley, New York, N.Y., United States of America.-   Baher et al. (1999) Anticancer Res 19:2917-2924.-   Baillie et al. (1995) Br J Cancer 72:257-267.-   Bass (2001) Nature 411:428-429.-   Batzer et al. (1991) Nucleic Acids Res 19:5081.-   Baumann et al. (1992) Intl J Radiat Oncol Biol Phys 23:803-809.-   Bauminger & Wilchek (1980) Methods Enzymol 70:151-159.-   Beaucage & Iyer (1993) Tetrahedron 49:1925-1963.-   Becerril et al. (1999) Biochem Biophys Res Commun 255:386-393.-   Beigelman et al. (1995) J Biol Chem 270:25702-25708.-   Bellon et al. (1997) Bioconjugate Chem 8:204-212.-   Betageri et al. (eds.) (1993) Liposome Drug Delivery Systems.    Technomic Publishing, Lancaster; Pa., United States of America.-   Blanchard et al. (1992) Mol Cell Biol 12:5373-5385.-   Brennan et al. (1998) Biotechnol Bioeng 61:33-45.-   Brown & Attardi (2005) Nat Rev Cancer 5:231-237.-   Burg et al. (1999) Cancer Res 59:2869-2874.-   Burgin et al. (1996) Biochemistry 35:14090-14097.-   Burlina et al. (1997) Bioorg Med Chem 5:1999-2010.-   Canadian Patent Application No. 2,359,180-   Cantley (2002) Science 296:1655-1657.-   Caruthers et al. (1992) Methods Enzymol 211:3-19.-   Chan et al. (1999) Annu Rev Biochem 68:965-1014.-   Chau et al. (2002) Oncogene 21:8817-8829.-   Chau et al. (2005) Am J Physiol Cell Physiol 289:C444-454.-   Chen et al. (2001) Nat Cell Biol 3:439-444.-   Cote et al. (2005) Nat Cell Biol 7:797-807.-   Cuneo et al. (2007) Cancer Res 67:4886-4893.-   Datta et al. (1999) Genes Dev 13:2905-2927.-   De Mesmaeker et al. (1994) in Carbohydrate Modifications in    Antisense Research, American Chemical Society, Washington, D.C.,    Symposium Series No. 580:24-39.-   Dent et al. (2003) Oncogene 22:5885-5896.-   Dracopoli et al. (eds.) (1997) Current Protocols in Human Genetics    on CD-ROM. John Wiley & Sons, New York, United States of America.-   Dudek et al. (1997) Science 275:661-665.-   Earnshaw & Gait (1998) Biopolymers 48:39-55.-   Ebert & Bunn (1998) Mol Cell Biol 18:4089-4096.-   Edwards et al. (2002) Cancer Res 62:4671-4677.-   Ekman et al. (2000) Oncogene 19:4151-4158.-   Elbashir et al. (2001a) Nature 411:494-498.-   Elbashir et al. (2001b) Genes Dev 15:188-200.-   Elbashir et al. (2001c) EMBO J 20:6877-6888.-   Ellerby et al. (1999) Nat Med 5:1032-1038.-   European Patent No. 0 439 095.-   Fang et al. (2000) Biochem J 352:135-143.-   Fewell et al. (2001) Mol Ther 3:574-583.-   Fire (1999) Trends Genet 15:358-363.-   Fire et al. (1998) Nature 391:806-811.-   Firth et al. (1995) J Biol Chem 270:21021-21027.-   Freier et al. (1986) Proc Natl Acad Sci U Glover & Hames, 1995-   Garcia-Barros et al. (2003) Science 300:1155-1159.-   GENBANK® Accession Nos. NM_(—)001109016; NM_(—)001721; NM_(—)009759;    NM_(—)203281; NP_(—)001102486; NP_(—)001712; NP_(—)033889;    NP_(—)975010; XM_(—)001101166; XM_(—)001101250; XM_(—)001101349;    XM_(—)001490091; XM_(—)548870; XM_(—)610012; XP_(—)001101166;    XP_(—)001101250; XP_(—)001101349; XP_(—)001490141; XP_(—)548870;    XP_(—)610012.-   Glover & Hames (1995) DNA Cloning: A Practical Approach, 2nd ed. IRL    Press at Oxford University Press, Oxford/N.Y., United States of    America.-   Goldman et al. (1997) Cancer Res 57:1447-1451.-   Gorski et al. (1999) Cancer Res 59:3374-3378.-   Greenberg et al. (1994) Mol Endocrinol 8:230-239.-   Gregoriadis (ed) (1993) Liposome Technology, 2nd ed. CRC Press, Boca    Raton, Fla., United States of America.-   Haimovitz-Friedman et al. (1994) J Exp Med 180:525-535.-   Hallahan & Virudachalam (1999) Radiat Res 152:6-13.-   Hallahan et al. (1995) Nat Med 1:786-791.-   Hallahan et al. (1996) Cancer Res 56:5150-5155.-   Hallahan et al. (1998) Cancer Res 58:5216-5220.-   Hallahan et al. (2001) J Control Release 74:183-191.-   Harlow & Lane (1988) Antibodies: A Laboratory Manual. Cold Spring    Harbor Laboratory Press, Cold Spring Harbor, N.Y., United States of    America.-   Hawiger & Timmons (1992) Methods Enzymol 215:228-243.-   Hawiger et al. (1989) Biochemistry 28:2909-2914.-   He et al. (2004) Cancer Biol Ther 3:96-101.-   Hilmas & Gillette (1975) Radiat Res 61:128-143.-   Hunziker & Leumann (1995) in Modern Synthetic Methods, VCH, Basel,    Switzerland 331-417.-   Ito et al. (1991) Cancer Res 51:255-260.-   Janoff (ed) (1999) Liposomes: Rational Design. M. Dekker, New York,    United States of America.-   Johnson (1976) Intl J Radiat Oncol Biol Phys 1:659-670.-   Jui et al. (2000) J Biol Chem 275:41124-41132.-   Kallman et al. (1972) Cancer Res 32:483-49.-   Karpeisky et al. (1998) Tetrahedron Lett 39:1131-1134.-   Katoh et al. (1995) Cancer Res 55:5687-5692.-   Kauffmann-Zeh et al. (1997) Nature 385:544-548.-   Kaukonen et al. (1996) Br J Haematol 94:455-460.-   Kawakami et al. (1999) Proc Natl Acad Sci USA 96:2227-2232.-   Kelley et al. (1999) J Biol Chem 274:26393-26398.-   Kim et al. (2002) J Biol Chem 277:30066-30071.-   Kirpotin et al. (1997) Biochemistry 36:66-75.-   Kurihara et al. (2000) J Clin Invest 106:763-771.-   Kyte & Doolittle (1982) J Mol Biol 157:105-132.-   Labat-Moleur et al. (1996) Gene Ther 3:1010-1017.-   Lasic & Martin (eds.) (1995) STEALTH® Liposomes. CRC Press, Boca    Raton, Fla., United States of America.-   Lee et al. (2000) Anticancer Res 20:417-422.-   Lee et al. (2001) Mol Cell Biol 21:8385-8397.-   Leibel & Phillips (2004) Textbook of Radiation Oncology, 2^(nd)    Edition, W.B. Saunders, Philadelphia, Pa., United States of America.-   Li et al. (1994) Leuk Lymphoma 13:65-70.-   Limbach et al. (1994) Nucleic Acids Res 22:2183-2196.-   Loakes (2001) Nucleic Acids Res 29:2437-2447.-   Mahajan et al. (1999) J Biol Chem 274:9587-9599.-   Mano (1999) Cytokine Growth Factor Rev 10:267-280.-   Manome et al. (1994) Cancer Res 54:5408-5413.-   Marin et al. (1997) Mol Med Today 3:396-403.-   Maruyama-Tabata et al. (2000) Gene Ther 7:53-60.-   Mesner et al. (1997) Adv Pharmacol 41:461-499.-   Miyagishi & Taira (2002) Nat Biotechnol 20:497-500.-   Neri et al. (1997) Nat Biotechnol 15:1271-1275.-   Nore et al. (2003) Biochim Biophys Acta 1645:123-132.-   Nykanen et al. (2001) Cell 107:309-321.-   Ohtsuka et al. (1985) J Biol Chem 260:2605-2608.-   Packer (1999) Arch Neurol 56:421-425.-   Pan et al. (2002) Mol Cell Biol 22:7512-7523.-   Pan et al. (2007) Chem Med Chem 2:58-61.-   Park et al. (1997) Cancer Lett 118:153-160.-   Pasqualini & Ruoslahti (1996) Nature 380:364-366.-   Pasqualini et al. (1997) Nat Biotechnol 15:542-546.-   Paz et al. (2005) Mol Cancer Ther 4:1801-1809.-   PCT International Patent Application Publication No. WO 91/03162; WO    92/07065; WO 92/07065; WO 93/15187; WO 93/23569; WO 97/26270; WO    98/10795; WO 98/13526; WO 99/07409; WO 99/32619; WO 99/54459; WO    00/01846; WO 00/44895; WO 00/44914; WO 00/63364; WO 01/04313; WO    01/29058; WO 01/36646; WO 01/68836; WO 01/75164; WO 01/92513; WO    02/044321; WO 02/055692.-   Qiu & Kung (2000) Oncogene 19:5651-5661.-   Perrault et al. (1990) Nature 344:565.-   Pieken et al. (1991) Science 253:314-317.-   Qiu et al. (1998) Proc Natl Acad Sci USA 95:3644-3649.-   Rossolini et al. (1994) Mol Cell Probes 8:91-98.-   Saltzman & Fung (1997) Adv Drug Deliv Rev 26:209-230.-   Sambrook & Russell (eds.) (2001) Molecular Cloning: A Laboratory    Manual (Third Edition), Cold Spring Harbor Laboratory Press, Cold    Spring Harbor, N.Y., United States of America.-   Scaringe et al. (1990) Nucleic Acids Res 18:5433-5441.-   Scharfmann et al. (1991) Proc Natl Acad Sci USA 88:4626-4630.-   Semenza & Wang (1992) Mol Cell Biol 12:5447-5454.-   Shabarova et al. (1991) Nature 359:843-845.-   Silhavy et al. (1984) Experiments with Gene Fusions. Cold Spring    Harbor Laboratory, Cold Spring Harbor, N.Y., United States of    America.-   Smith et al. (2001) Bioessays 23:436-446.-   Somervaille et al. (2001) Blood 98:1374-1381.-   Song et al. (1972) Radiology 104:693-697.-   Sonveaux et al. (2007) Int J Radiat Oncol Biol Phys 67:1155-1162.-   Staba et al. (1998) Gene Ther 5:293-300.-   Taghian et al. (1993) Intl J Radiat Oncol Biol Phys 25:243-249.-   Tam et al. (2000) Gene Ther 7:1867-1874.-   Tan & Hallahan (2003) Cancer Res 63:7663-7667.-   Tan et al. (2006) Cancer Res 66:2320-2327.-   Ting et al. (1991) Intl J Radiat Biol 60:335-339.-   Tomlinson et al. (2004) J Biol Chem 279:55089-55096.-   Turner et al. (1987) Cold Spring Harb Symp Quant Biol LII:123-133.-   U.S. Patent Application Publication No. 20030175703.-   U.S. Pat. Nos. 4,196,265; 4,235,871; 4,551,482; 4,554,101;    4,946,778; 5,011,634; 5,091,513; 5,111,867; 5,132,405; 5,260,203;    5,270,163; 5,334,711; 5,490,840; 5,510,103; 5,567,588; 5,574,172;    5,627,053; 5,632,991; 5,651,991; 5,667,988; 5,672,695; 5,677,427;    5,683,867; 5,688,931; 5,702,892; 5,714,166; 5,716,824; 5,780,225;    5,786,387; 5,840,479; 5,849,877; 5,854,027; 5,854,038; 5,855,900;    5,858,410; 5,892,019; 5,922,254; 5,922,356; 5,922,545; 5,948,647;    5,948,767; 5,985,279; 5,994,392; 5,998,203; 6,001,311; 6,054,561;    6,056,938; 6,057,098; 6,071,890; 6,090,925; 6,106,866; 6,120,787;    6,127,339; 6,132,766; 6,174,708; 6,180,084; 6,190,700; 6,197,333;    6,200,598; 6,210,707; 6,217,886; 6,221,958; 6,238,704; 6,238,705;    6,245,740; 6,248,878; 6,262,127; 6,267,981; 6,287,587; 6,296,832;    6,296,842; 6,300,074; 6,312,713; 6,335,035; 6,506,559; 6,706,482;    6,855,496; 7,067,649; 7,176,295.-   Uckun et al. (2002) Clin Cancer Res 8:1224-1233.-   Uhlman & Peyman (1990) Chem Rev 90:543-549.-   Usman & Cedergren (1992) Trends Biochem Sci 17:334-339.-   Usman et al. (1987) J Am Chem Soc 109: 7845-7854.-   Usman et al. (1994) Nucleic Acids Symp Ser 31:163-164.-   Usman et al. (1996) Curr Opin Struct Biol 6:527-533.-   Valerie et al. (2007) Mol Cancer Ther 6:789-801.-   Vargas et al. (2002) J Biol Chem 277:9351-9357.-   Verma & Eckstein (1998) Annu Rev Biochem 67:99-134.-   Walker et al. (1980) N Engl J Med 303:1323-1329.-   Wallner et al. (1989) Intl J Radiat Oncol Biol Phys 16:1405-1409.-   Weiner & Chun (1999) Proc Natl Acad Sci USA 96:5233-5238.-   Wen et al. (1999) J Biol Chem 274:38204-38210.-   Wianny & Zernicka-Goetz (1999) Nature Cell Biol 2:70-75.-   Williams et al. (1993) J Clin Invest 92:503-508.-   Wincott & Usman (1997) Methods Mol Bio 74: 59-68.-   Wincott et al. (1995) Nucleic Acids Res 23:2677-2684.-   Wymann & Pirola (1998) Biochim Biophys Acta 1436:127-150.-   Yacoub et al. (2006) Endocr Relat Cancer 13 Suppl 1:S99-S114.-   Yamaura et al. (1976) Intl J Radiat Biol Relat Stud Phys Chem Med    30:179-187.-   Yang et al. (2002) J Biol Chem 277:30219-30226.-   Yao & Cooper (1995) Science 267:2003-2006.-   Yu et al. (1999) Cancer Res 59:4200-4203.-   Zamore et al. (2000) Cell 101:25-33.-   Zhang et al. (2003) J Biol Chem 278:51267-51276.-   Zingg et al. (2004) Cancer Res 64:5398-5406.

It will be understood that various details of the described subjectmatter can be changed without departing from the scope of the describedsubject matter. Furthermore, the foregoing description is for thepurpose of illustration only, and not for the purpose of limitation.

What is claimed is:
 1. A method for inhibiting proliferation of a tumorin a subject, the method comprising: (a) administering to the subject aneffective amount of an inhibitor of a biological activity of a bonemarrow X kinase (Bmx) gene product; and (b) irradiating vasculaturesupplying blood flow to the tumor, wherein the vasculature supplyingblood flow to the tumor expresses the Bmx gene product and wherebyproliferation of the tumor in the subject is inhibited.
 2. The method ofclaim 1, wherein the subject is a mammal.
 3. The method of claim 2,wherein the Bmx gene product: (a) is encoded by a naturally occurringnucleic acid sequence that is at least 95% identical to nucleotides174-2198 of SEQ ID NO: 1; or (b) is encoded by a naturally occurringnucleic acid sequence that is at least 95% identical to nucleotides112-2136 of SEQ ID NO:
 3. 4. The method of claim 1, wherein theinhibitor is selected from the group consisting of(2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, an antibodythat specifically binds to the Bmx gene product to inhibit a biologicalactivity of the Bmx gene product, and a nucleic acid that inhibits abiological activity of the Bmx gene product by RNA interference.
 5. Themethod of claim 4, wherein the nucleic acid that inhibits a biologicalactivity of the Bmx gene product by RNA interference comprises a shortinterfering RNA (siRNA) or a short hairpin RNA (shRNA) that targets aBmx gene product encoded by a nucleic acid sequence comprisingnucleotides 174-2198 of SEQ ID NO: 1 or nucleotides 112-2136 of SEQ IDNO:
 3. 6. The method of claim 5, wherein the siRNA or the shRNA isencoded by a recombinant virus and the administering comprisesadministering an effective amount of the recombinant virus to thesubject to modulate proliferation of a cell or of a tissue in thesubject.
 7. A method for increasing the radiosensitivity of a tumor, themethod comprising: (a) contacting a cell of vasculature supplying bloodflow to the tumor with an effective amount of an inhibitor of abiological activity of a bone marrow X kinase (Bmx) gene product; and(b) irradiating the cell of the vasculature supplying blood flow to thetumor, whereby the radiosensitivity of the tumor is increased.
 8. Themethod of claim 7, wherein the inhibitor of a biological activity of aBmx gene product comprises a bone marrow X kinase (Bmx) antagonist, avector encoding a bone marrow X kinase (Bmx) antagonist, or acombination thereof.
 9. The method of claim 7, wherein the tumor is aradiation resistant tumor.
 10. The method of claim 7, wherein thesubject is a mammal.
 11. The method of claim 7, wherein the tumor ispresent within a subject and the method comprises administering a bonemarrow X kinase (Bmx) antagonist to the subject.
 12. The method of claim11, wherein the administering comprises administering a compositioncomprising: (a) a bone marrow X kinase (Bmx) antagonist, a vectorencoding a bone marrow X kinase (Bmx) antagonist, or a combinationthereof; and (b) a pharmaceutically acceptable carrier.
 13. The methodof claim 12, wherein the bone marrow X kinase (Bmx) antagonist isselected from the group consisting of(2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, an antibodythat specifically binds to the Bmx gene product to inhibit a biologicalactivity of the Bmx gene product, and a nucleic acid that inhibits abiological activity of the Bmx gene product by RNA interference.
 14. Themethod of claim 7, wherein the bone marrow X kinase (Bmx) antagonistcomprises a small interfering RNA (siRNA) targeted to a Bmx geneproduct.
 15. A method for suppressing tumor growth in a subject, themethod comprising: (a) administering to the subject an effective amountof an inhibitor of a biological activity of a bone marrow X kinase (Bmx)gene product; and (b) treating vasculature supplying blood flow to thetumor with ionizing radiation, whereby tumor growth is suppressed. 16.The method of claim 15, wherein the subject is a mammal.
 17. The methodof claim 15, wherein the administering comprises administering aminimally therapeutic dose of the inhibitor.
 18. The method of claim 15,wherein the administering comprises administering a compositioncomprising: (a) a bone marrow X kinase (Bmx) antagonist, a vectorencoding a bone marrow X kinase (Bmx) antagonist, or a combinationthereof; and (b) a pharmaceutically acceptable carrier.
 19. The methodof claim 15, wherein the inhibitor is selected from the group consistingof (2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, anantibody that specifically binds to the Bmx gene product to inhibit abiological activity of the Bmx gene product, and a nucleic acid thatinhibits a biological activity of the Bmx gene product by RNAinterference.
 20. The method of claim 15, wherein the inhibitorcomprises a small interfering RNA (siRNA) targeted to a Bmx geneproduct.
 21. The method of claim 15, wherein the tumor is a radiationresistant tumor.
 22. The method of claim 15, wherein the treatingvasculature supplying blood flow to the tumor with ionizing radiationcomprises treating vasculature supplying blood flow to the tumor with asubtherapeutic dose of ionizing radiation.
 23. A method for inhibitingtumor blood vessel growth in a subject, the method comprising: (a)administering to the subject an effective amount of an inhibitor of abiological activity of a bone marrow X kinase (Bmx) gene product; and(b) treating vasculature supplying blood flow to the tumor with ionizingradiation, whereby tumor blood vessel growth is inhibited.
 24. Themethod of claim 23, wherein the administering comprises administering aminimally therapeutic dose of the inhibitor.
 25. The method of claim 23,wherein the inhibitor comprises a composition comprising: (a) a bonemarrow X kinase (Bmx) antagonist, a vector encoding a bone marrow Xkinase (Bmx) antagonist, or a combination thereof; and (b) apharmaceutically acceptable carrier.
 26. The method of claim 23, whereinthe inhibitor is selected from the group consisting of(2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, an antibodythat specifically binds to the Bmx gene product to inhibit a biologicalactivity of the Bmx gene product, and a nucleic acid that inhibits abiological activity of the Bmx gene product by RNA interference.
 27. Themethod of claim 26, wherein the inhibitor comprises a small interferingRNA (siRNA) targeted to a Bmx gene product.
 28. The method of claim 23,wherein the subject is a mammal.
 29. The method of claim 23, wherein thetumor is a radiation resistant tumor.
 30. The method of claim 23,wherein the treating vasculature supplying blood flow to the tumor withionizing radiation comprises treating vasculature supplying blood flowto the tumor with a subtherapeutic dose of ionizing radiation.
 31. Themethod of claim 23, further comprising reducing the vascular lengthdensity of tumor blood vessels present within the vasculature supplyingblood flow to the tumor.
 32. A method for inhibiting a conditionassociated with undesirable angiogenesis in a subject, the methodcomprising: (a) administering to the subject an effective amount of abone marrow X kinase (Bmx) antagonist; and (b) irradiating a site ofundesirable angiogenesis in the subject, wherein the site of undesirableangiogenesis comprises vascular endothelium cells that express a Bmxgene product, whereby a condition associated with undesirableangiogenesis in the subject is inhibited.
 33. The method of claim 32,wherein the condition associated with undesirable angiogenesis isselected from the group consisting of a cancer, a tumor, maculardegeneration, and endometriosis.
 34. The method of claim 32, wherein theBmx antagonist is selected from the group consisting of(2Z)-2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, an antibodythat specifically binds to the Bmx gene product to inhibit a biologicalactivity of the Bmx gene product, and a nucleic acid that inhibits abiological activity of the Bmx gene product by RNA interference.
 35. Themethod of claim 34, wherein the nucleic acid that inhibits a biologicalactivity of the Bmx gene product by RNA interference comprises a smallinterfering RNA (siRNA) targeted to a Bmx gene product.
 36. The methodof claim 32, wherein the subject is a mammal.
 37. The method of claim11, wherein the administering comprises administering a minimallytherapeutic dose of the Bmx antagonist to the subject.